Aerosol fluoroquinolone formulations for improved pharmacokinetics

ABSTRACT

The present invention relates to the field of antimicrobial agents. In particular, the present invention relates to the use of aerosolized fluoroquinolones formulated with divalent or trivalent cations and having improved pulmonary availability for the treatment and management of bacterial infections of the lung and upper respiratory tract.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/132,122 filed Apr. 18, 2016, allowed, which is a continuation of U.S.application Ser. No. 14/333,583 filed Jul. 17, 2014, issued as U.S. Pat.No. 9,326,936, which is a continuation of U.S. application Ser. No.12/574,680 filed Oct. 6, 2009, issued as U.S. Pat. No. 8,815,838, whichclaims priority to U.S. Application No. 61/103,501 filed Oct. 7, 2008,which is hereby expressly incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of antimicrobial agents. Inparticular, the present invention relates to the use of aerosolizedfluoroquinolones formulated with divalent or trivalent cations withimproved pulmonary availability for the treatment and management ofbacterial infections of the lung and upper respiratory tract.

BACKGROUND

Gram-negative bacteria are intrinsically more resistant to antibioticsthan gram-positive bacteria due to the presence of a second outermembrane, which provides an efficient barrier to both hydrophilic andhydrophobic compounds. Consequently, there are few classes ofantibiotics available to treat Gram-negative infections. Indeed, onlyseveral representatives of beta-lactams, aminoglycosides andfluoroquinolones have in vitro antibacterial activity againstPseudomonas aeruginosa and have been shown to have clinical utility, notsurprisingly, development of resistance to such antibiotics iswell-documented.

Respiratory diseases afflict millions of people across the world leadingto suffering, economic loss and premature death, including infections ofacute, subacute and chronic duration of the nasal cavity or four sinuses(each which have left and right halves, the frontal, the maxillary theethmoid and the sphenoid), or the larynx, trachea or lung (bronchi,bronchioles, alveoli).

Pulmonary infections caused by gram-negative bacteria represent aparticular challenge. Causative agents are usually found in sputum,pulmonary epithelial lining fluid, alveolar macrophages and bronchialmucosa. Acute exacerbations of pulmonary infection, periodicallyobserved in patients with cystic fibrosis, COPD, chronic bronchitis,bronchiectasis, acute and chronic pneumonias, and many other pulmonaryinfections. Prevention of these exacerbations as well as their treatmentis often difficult especially when highly resistant pathogens such asPseudomonas aeruginosa and Burkholderia cepacia complex are involved.For most treatment protocols, high doses are required to maintaineffective concentrations at the site of infection. In the case ofaminoglycosides, nephrotoxicity and ototoxicity are also directlyrelated to prolonged elevations of serum antibiotic concentrations. Inan attempt to achieve an optimal outcome for the patient, cliniciansroutinely use a combination of two or more antibiotics such asceftazidime and tobramycin, which are administered at high doses for 2weeks, with the aim of achieving antibiotic synergy (J. G. denHollander, et al., “Synergism between tobramycin and ceftazidime againsta resistant Pseudomonas aeruginosa strain, tested in an in vitropharmacokinetic model” Antimicrob. Agents Chemother. (1997), 41,95-100). For example, successful treatments require that ceftazidime beadministered either every 8 hours or by continuous infusion to maximizethe time that the serum concentration is above the minimum inhibitoryconcentration (M. Cazzola, et al., “Delivering antibacterials to thelungs: Considerations for optimizing outcomes” Am. J. Respir. Med.(2002), 1, 261-272).

Aerosol administration of antibiotics directly to the site of infection,ensuring high local concentrations coupled with low systemic exposurerepresent an attractive alternative for the treatment of pulmonaryinfections. Aerosolized tobramycin is used for treatment of pseudomonalbacterial infections in patients with cystic fibrosis. The rationalebehind this technique is to administer the drug directly to the site ofinfection and thereby alleviate the need to produce high serumconcentrations by the standard intravenous method. An advantageofaerosol administration is that many patients can self-administer theantibiotic, and this treatment method may negate the need for lengthyhospitalization (M. E. Hodson “Antibiotic treatment: Aerosol therapy”,Chest (1988), 94, 156S-160S; and M. S. Zach “Antibiotic aerosoltreatment” Chest (1988), 94, 160S-162S). However, tobramycin iscurrently the only FDA-approved aerosol antibiotic in the United States.And while it continues to play an important role in the management ofrecurrent infections in cystic fibrosis patients, its clinical utilityis inadvertently being diminished due to development of resistance. Inaddition, the impact of total high concentrations achieved after aerosoladministration is being somewhat diminished due to high binding oftobramycin to the components of cystic fibrosis sputum. Thus, there is aneed for improved aerosolized antibiotics.

SUMMARY

The present invention relates to the use of aerosolized fluoroquinolonesformulated with divalent or trivalent cations having improved pulmonaryavailability for the treatment and management of bacterial infections ofthe lung and upper respiratory tract. Some methods include treating apulmonary infection including administering to a subject in needthereof, an effective amount of an aerosol solution of levofloxacin orofloxacin in combination with a divalent or trivalent cation withimproved pulmonary availability and exposure to levofloxacin orofloxacin.

Methods for treating a pulmonary infection are provided. Some suchmethods include administering to a human having a pulmonary infection anaerosol of a solution comprising levofloxacin or ofloxacin and adivalent or trivalent cation to achieve a maximum lung sputumconcentration (C_(max)) of at least 1200 mg/L and a lung sputum areaunder the curve (AUC) of at least 1500 h·mg/L. In more embodiments,methods for treating a chronic lung infection are provided. Some suchmethods can include administering to a subject having a chronic lunginfection an aerosol of a solution comprising levofloxacin or ofloxacinand a divalent or trivalent cation. In more embodiments, pharmaceuticalcompositions are provided. Some such compositions can include an aqueoussolution consisting essentially of from 80 mg/ml to 120 mg/mllevofloxacin or ofloxacin and from 160 mM to 220 mM of a divalent ortrivalent cation, wherein the solution has a pH from 5 to 7 and anosmolality from 300 mOsmol/kg to 500 mOsmol/kg.

Some embodiments include methods for treating a pulmonary infection thatinclude administering to a human having said pulmonary infection anaerosol of a solution that includes levofloxacin or ofloxacin and adivalent or trivalent cation to achieve a maximum lung sputumconcentration (C_(max)) of at least about 1200 mg/L and a lung sputumarea under the curve (AUC) of at least about 1500 h·mg/L.

Some embodiments include methods of treating a chronic lung infectionthat include administering to a subject having a chronic lung infectionan aerosol of a solution that includes levofloxacin or ofloxacin and adivalent or trivalent cation.

Some embodiments include pharmaceutical compositions that include anaqueous solution consisting essentially of from about 80 mg/ml to about120 mg/ml levofloxacin or ofloxacin and from about 160 mM to about 240mM of a divalent or trivalent cation, wherein the solution has a pH fromabout 5 to about 7 and an osmolality from about 300 mOsmol/kg to about500 mOsmol/kg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of plasma levofloxacin concentration in rats afterintravenous administration, aerosol administration of levofloxacin (LVX)formulated in saline, or aerosol administration of levofloxacinformulated with MgCl₂.

FIG. 2 shows a graph of recovery of levofloxacin from lung homogenate inrats after a single 10 mg/kg dose of aerosol levofloxacin formulatedwith saline, Ca⁺², Mg⁺² or Zn²⁺.

FIG. 3 shows a schematic of a pharmacokinetic model for deconvolution ofserum levofloxacin concentrations following aerosol administration of adrug.

FIG. 4 shows a graph of plasma levofloxacin (LVX) concentrations afteradministration of 50 mg LVX in saline by aerosol or intravenous routes.

FIG. 5 shows a graph of the estimated amount of drug remaining in thelung compartment following aerosol administration of 50 mg respirabledrug dose levofloxacin in saline, where amount of drug remaining isestimated using deconvolution of serum data.

FIG. 6 shows a graph of serum levofloxacin concentrations in normalhealthy volunteers and cystic fibrosis patients after a singleintravenous or aerosol dose of levofloxacin administered in saline.Doses are shown as estimated RDD; the 20 mg and 40 mg RDDs representnebulizer loaded doses of 43.3 and 86.6 mg, respectively.

FIG. 7 shows a graph of deconvolution estimates of the average amount oflevofloxacin remaining in the lung in 7 healthy volunteers and 9 CFpatients following a single aerosol dose of 43.3 mg loaded intonebulizer (estimated respirable delivered dose (RDD) of 20 mg, and a86.6 mg dose loaded into the nebulizer (estimated RDD of 40 mg)formulated in normal saline.

FIG. 8A shows a graph of the estimated levofloxacin concentrations inlung epithelial lining fluid following a single 20 mg respirable drugdose (43.3 mg of levofloxacin loaded into the nebulizer) formulated innormal saline in CF patients. FIG. 8B shows a graph of the estimatedlevofloxacin concentrations in lung epithelial lining fluid following asingle 40 mg respirable drug dose (86.6 mg loaded into the nebulizer)formulated in normal saline in CF patients.

FIG. 9 shows a graph of sputum levofloxacin concentrations in CFsubjects following a single IV infusion or aerosol dose of levofloxacinformulated in normal saline. Doses are shown as estimated RDD; the 20 mgand 40 mg RDDs represent nebulizer loaded doses of 43.3 and 86.6 mg,respectively.

FIG. 10 shows a graph depicting modeled sputum levofloxacinconcentrations in CF subjects following various doses and routes ofadministration (aerosol doses formulated in normal saline) oflevofloxacin.

FIG. 11 shows a graph of sputum levofloxacin (LVX) concentration incystic fibrosis patients, after aerosol administration of 50 mg/ml LVXformulated with MgCl₂ or with saline using an estimated 40 mg respirabledrug dose (RDD) which corresponds to a 86.6 mg loaded drug dose.

FIG. 12 shows a graph of the arithmetic mean serum concentrations oflevofloxacin in cystic fibrosis patients, after aerosol administrationof a 180 mg dose with 50 mg/ml or 100 mg/ml levofloxacin solution, or a240 mg dose with a 100 mg/ml levofloxacin solution. Treatment was oncedaily for 7 days. The 50 mg/ml levofloxacin formulation contained 100 mMmagnesium chloride and 150 mM lactose, and the 100 mg/ml levofloxacincontained 200 mM magnesium chloride and no lactose.

FIG. 13 shows a graph of the arithmetic mean sputum concentrations oflevofloxacin in cystic fibrosis patients, after aerosol administrationof a 180 mg dose with 50 mg/ml or 100 mg/ml levofloxacin solution, or a240 mg dose with a 100 mg/ml levofloxacin solution. Treatment was oncedaily for 7 days. The 50 mg/ml levofloxacin formulation contained 100 mMmagnesium chloride and 150 mM lactose, and the 100 mg/ml levofloxacincontained 200 mM magnesium chloride and no lactose.

FIG. 14 shows a graph of mean sputum levofloxacin levels in cysticfibrosis patients following a single nebulized dose of levofloxacinformulated in saline compared to formulation in a solution of magnesiumchloride. Both formulations were nebulized using a Pari eFlow nebulizerusing vibrating mesh technology with the same mesh head design and poresize. The nebulizer loaded dose of levofloxacin in saline was 87 mg andthe nebulizer loaded dose of levofloxacin was 180 mg for the formulationusing magnesium chloride. Data were normalized to an 87 mg dose bymultiplying the observed sputum levofloxacin concentrations obtainedwith the magnesium chloride formulation by 87/180 (0.48).

FIG. 15 shows a graph of the change in log colony forming units (CFU) ofK. pneumoniae ATCC 43816/lung in mice, after aerosol administration of10 mg/kg or 20 mg/kg doses of levofloxacin (LVX) formulated with andwithout MgCl₂.

FIG. 16 shows a graph of the log colony forming units (CFU) of P.aeruginosa ATCC 27853/lung in a mouse acute lung infection model, afteraerosol administration of 125 mg/kg, 63 mg/kg, or 32 mg/kg levofloxacin(LVX) with MgCl₂, or intraperitoneal administration (IP) of 125 mg/kg,63 mg/kg, or 32 mg/kg levofloxacin. Values shown are mean±SD logCFU/lung. Treatment groups (n=8) received 2 doses of antibiotics over 24h. (p<0.05 for comparisons of aerosol vs. IP administration for eachdose).

FIG. 17 shows a graph of log colony forming units (CFU) of P. aeruginosaNH57388A/lung in a murine chronic lung infection model, after twicedaily aerosol administration of 60 mg/kg, 30 mg/kg, or 15 mg/kglevofloxacin (LVX) with MgCl₂, or twice daily intraperitonealadministration of 60 mg/kg, 30 mg/kg, or 15 mg/kg levofloxacin. Aerosoldoses of levofloxacin were formulated in magnesium chloride. Valuesshown are mean±SD log CFU/lung. Treatment groups (n=8) received 2 dosesof antibiotics per day for 72 h. (p<0.05 for comparison of aerosol vsintraperitoneal for the same dose).

FIG. 18 shows a graph of log colony forming units (CFU) of P. aeruginosaATCC 27853/lung in a murine acute lethal lung infection model, aftertwice daily aerosol administration of 60 mg/kg of levofloxacin, 60 mg/kgtobramycin, or 400 mg/kg aztreonam, or once daily aerosol administrationof 120 mg/kg levofloxacin. Aerosol doses of levofloxacin were formulatedin magnesium chloride. Treatment groups (n=8) received drug for 48 h.Values shown are mean±SD log CFU/lung. (p<0.05).

FIG. 19 shows a graph of the percent survival over time of mice infectedwith P. aeruginosa ATCC 27853 in a murine lethal lung infection model,and treated with twice daily aerosol administration of 60 mg/kg oflevofloxacin, 60 mg/kg tobramycin, or 400 mg/kg aztreonam, or once dailyaerosol administration of 120 mg/kg levofloxacin. Aerosol doses oflevofloxacin were formulated in magnesium chloride. Treatment groups ofmice (n=10) received drug for 48 h. Survival was monitored through day 9following infection.

FIG. 20 shows a graph of the log colony forming units (CFU) of P.aeruginosa NH57388A/lung in a murine chronic lung infection model, aftertwice daily aerosol administration of 60 mg/kg of levofloxacin, 60 mg/kgtobramycin, or 400 mg/kg aztreonam. Treatment groups were treated twicea day for three consecutive days. Aerosol doses of levofloxacin wereformulated in magnesium chloride. Values shown are mean±SD log CFU/lung.Aerosol levofloxacin resulted in lower bacterial counts than aztreonamor untreated control mice (p<0.05).

DETAILED DESCRIPTION

The present invention relates to the field of antimicrobial agents. Inparticular, the present invention relates to the use of aerosolizedfluoroquinolones formulated with divalent or trivalent cations havingimproved pulmonary availability and thus better bactericidal activityfor the treatment and management of bacterial infections of the lung andupper respiratory tract.

Many of the problems associated with antimicrobial-resistant pathogenscould be alleviated if the concentration of the antimicrobial could besafely increased at the site of infection. For example, pulmonaryinfections may be treated by administration of the antimicrobial agent,at high concentrations directly to the site of infection withoutincurring large systemic concentrations of the antimicrobial.Accordingly, some embodiments disclosed herein are improved methods fordelivering drug compositions to treat pulmonary bacterial infections.More specifically, described herein are formulations of fluoroquinoloneswith divalent or trivalent cations that achieve a desirablepharmacokinetic profile of the fluoroquinolone in humans beneficial forincreasing efficacy and reducing the emergence of drug resistance.

Accordingly, some embodiments described herein include methods andcompositions that include fluoroquinolones where absorption from lungtissue or the upper airway into systemic circulation after aerosol isretarded. In some such embodiments, fluoroquinolones are complexed withdivalent cations in a manner that does not significantly diminish theirantimicrobial activity. Such complexes may be for the treatment,maintenance or prevention of infection. In addition, such complexes canshow higher concentrations of drug at the sites of infection (e.g., theupper and/or lower respiratory system), and higher efficacy, compared toa fluoroquinolone not combined with divalent or trivalent cations.

Some embodiments of the present invention relate to methods for treatinga pulmonary infection, and compositions of levofloxacin or ofloxacinformulated with a divalent or trivalent cation. It has been discoveredthat particular methods and compositions described herein achieve animproved availability of levofloxacin or ofloxacin in the lungs ofsubjects. An increased availability in the lungs of antimicrobial agentsis useful in the treatment of pulmonary infections, and is particularlyadvantageous in the treatment of conditions such as cystic fibrosis andchronic obstructive pulmonary disorders, including for example, chronicbronchitis, bronchiectasis, and some asthmas.

Improved availability in the lung can be indicated using a variety ofpharmodynamic-pharmacokinetic parameters relating to factors such asincreased concentration of a drug in the lung and/or length of time adrug is retained in the lung. Such factors can include lung sputum areaunder curve (AUC), and maximum lung sputum concentration (C_(max)).

Typically, administering aerosolized antimicrobial agents to the lungscan provide high concentrations in the lungs without incurring highsystemic concentrations. However, the methods and compositions providedherein achieve unexpectedly increased availability in the lungs.

Generally, the compositions provided herein can comprise solutions oflevofloxacin or ofloxacin formulated with a divalent or trivalentcation, such as Mg′. In some embodiments, the compositions can lack aparticular excipient, such as lactose. The compositions may beadministered using devices such as nebulizers or a microspray aerosoldevice inserted directly into the trachea of animals, and can be used totreat a wide variety of bacteria. In addition, methods and compositionsprovided herein can include additional active agents useful in thetreatment of pulmonary infections, and disorders associated withpulmonary infections, such as cystic fibrosis, and chronic obstructivepulmonary disease, including chronic bronchitis and some asthmas.

Definitions

The term “administration” or “administering” refers to a method ofgiving a dosage of an antimicrobial pharmaceutical composition to avertebrate. The preferred method of administration can vary depending onvarious factors, e.g., the components of the pharmaceutical composition,the site of the potential or actual bacterial infection, the microbeinvolved, and the severity of an actual microbial infection.

A “carrier” or “excipient” is a compound or material used to facilitateadministration of the compound, for example, to increase the solubilityof the compound. Solid carriers include, e.g., starch, lactose,dicalcium phosphate, sucrose, and kaolin. Liquid carriers include, e.g.,sterile water, saline, buffers, non-ionic surfactants, and edible oilssuch as oil, peanut and sesame oils. In addition, various adjuvants suchas are commonly used in the art may be included. These and other suchcompounds are described in the literature, e.g., in the Merck Index,Merck & Company, Rahway, N.J. Considerations for the inclusion ofvarious components in pharmaceutical compositions are described, e.g.,in Gilman et al. (Eds.) (1990); Goodman and Gilman's: ThePharmacological Basis of Therapeutics, 8th Ed., Pergamon Press,incorporated by reference herein in its entirety.

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes humans, cattle, horses, dogs, and cats, but alsoincludes many other species.

The term “microbial infection” refers to the undesired proliferation orpresence of invasion of pathogenic microbes in a host organism. Thisincludes the excessive growth of microbes that are normally present inor on the body of a mammal or other organism. More generally, amicrobial infection can be any situation in which the presence of amicrobial population(s) is damaging to a host mammal. Thus, a microbialinfection exists when excessive numbers of a microbial population arepresent in or on a mammal's body, or when the effects of the presence ofa microbial population(s) is damaging the cells or other tissue of amammal.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of the compounds of thisinvention and, which are not biologically or otherwise undesirable. Inmany cases, the compounds of this invention are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto. Pharmaceutically acceptable acidaddition salts can be formed with inorganic acids and organic acids.Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids from which salts can bederived include, for example, acetic acid, propionic acid, naphtoicacid, oleic acid, palmitic acid, pamoic (emboic) acid, stearic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, ascorbic acid,glucoheptonic acid, glucuronic acid, lactic acid, lactobioic acid,tartaric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like. Pharmaceutically acceptable base additionsalts can be formed with inorganic and organic bases. Inorganic basesfrom which salts can be derived include, for example, sodium, potassium,lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese,aluminum, and the like; particularly preferred are the ammonium,potassium, sodium, calcium and magnesium salts. Organic bases from whichsalts can be derived include, for example, primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines, basic ion exchange resins, and thelike, specifically such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, histidine, arginine, lysine, benethamine,N-methyl-glucamine, and ethanolamine. Other acids include dodecylsufuricacid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, andsaccharin.

“Solvate” refers to the compound formed by the interaction of a solventand fluoroquinolone antimicrobial, a metabolite, or salt thereof.Suitable solvates are pharmaceutically acceptable solvates includinghydrates.

In the context of the response of a microbe, such as a bacterium, to anantimicrobial agent, the term “susceptibility” refers to the sensitivityof the microbe for the presence of the antimicrobial agent. So, toincrease the susceptibility means that the microbe will be inhibited bya lower concentration of the antimicrobial agent in the mediumsurrounding the microbial cells. This is equivalent to saying that themicrobe is more sensitive to the antimicrobial agent. In most cases theminimum inhibitory concentration (MIC) of that antimicrobial agent willhave been reduced. The MIC₉₀ can include the concentration to inhibitgrowth in 90% of organisms.

By “therapeutically effective amount” or “pharmaceutically effectiveamount” is meant a fluoroquinolone antimicrobial agent, as disclosed forthis invention, which has a therapeutic effect. The doses offluoroquinolone antimicrobial agent which are useful in treatment aretherapeutically effective amounts. Thus, as used herein, atherapeutically effective amount means those amounts of fluoroquinoloneantimicrobial agent which produce the desired therapeutic effect asjudged by clinical trial results and/or model animal infection studies.In particular embodiments, the fluoroquinolone antimicrobial agent areadministered in a predetermined dose, and thus a therapeuticallyeffective amount would be an amount of the dose administered. Thisamount and the amount of the fluoroquinolone antimicrobial agent can beroutinely determined by one of skill in the art, and will vary,depending on several factors, such as the particular microbial straininvolved. This amount can further depend upon the patient's height,weight, sex, age and medical history. For prophylactic treatments, atherapeutically effective amount is that amount which would be effectiveto prevent a microbial infection.

A “therapeutic effect” relieves, to some extent, one or more of thesymptoms of the infection, and includes curing an infection. “Curing”means that the symptoms of active infection are eliminated, includingthe total or substantial elimination of excessive members of viablemicrobe of those involved in the infection to a point at or below thethreshold of detection by traditional measurements. However, certainlong-term or permanent effects of the acute or chronic infection mayexist even after a cure is obtained (such as extensive tissue damage).As used herein, a “therapeutic effect” is defined as a statisticallysignificant reduction in bacterial load in a host, emergence ofresistance, pulmonary function, or improvement in infection symptoms orfunctional status as measured by human clinical results or animalstudies.

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a pharmaceutical composition for prophylactic and/ortherapeutic purposes. The term “prophylactic treatment” refers totreating a patient who is not yet infected, but who is susceptible to,or otherwise at risk of, a particular infection such that there is areduced onset of infection. The term “therapeutic treatment” refers toadministering treatment to a patient already suffering from an infectionthat may be acute or chronic. Treatment may eliminate the pathogen, orit may reduce the pathogen load in the tissues that results inimprovements measured by patients symptoms or measures of lung function.Thus, in preferred embodiments, treating is the administration to amammal (either for therapeutic or prophylactic purposes) oftherapeutically effective amounts of a fluoroquinolone antimicrobialagent.

Pharmacokinetics (PK) is concerned with the time course of antimicrobialconcentration in the body. Pharmacodynamics (PD) is concerned with therelationship between pharmacokinetics and the antimicrobial efficacy invivo. PK/PD parameters correlate antimicrobial exposure withantimicrobial activity. The rate of killing by antimicrobial isdependent on antimicrobial mode of action and is determined by eitherthe length of time necessary to kill (time-dependent) or the effect ofincreasing concentrations alone (concentration-dependent) or integratedover time as an area under the concentration-time curve (AUC). Topredict the therapeutic efficacy of antimicrobials with diversemechanisms of action different PK/PD parameters may be used. PK/PDparameters may be used to determine the availability of antimicrobialcompositions, for example, availability of a antimicrobial agent in acomposition in the pulmonary system, and/or bioavailability of aantimicrobial agent in a composition in plasma/serum.

“AUC/MIC ratio” is one example of a PK/PD parameter. AUC is defined asthe area under the plasma/serum or site-of-infection concentration-timecurve of an antimicrobial agent in vivo (in animal or human). Forexample, the site of infection and/or the site where concentration ismeasured can include portions of the pulmonary system, such as bronchialfluid and/or sputum. Accordingly, AUC may be a serum AUC, or a pulmonaryAUC based on concentrations in serum and pulmonary tissues (sputum,epithelial lining fluid, or homogenates of whole tissue). AUC₍₀₋₄₎ caninclude the area under curve for time zero to a specific time ‘t.’AUC_((0-inf)) can include the area under curve from time zero toinfinity. AUC/MIC ratio is determined by dividing the 24-hour-AUC for anindividual antimicrobial by the MIC for the same antimicrobialdetermined in vitro. Activity of antimicrobials with the dose-dependentkilling (such as fluoroquinolones) is well predicted by the magnitude ofthe AUC/MIC ratio. The AUC:MIC ratio can also prevent selection ofdrug-resistant bacteria.

“C_(max):MIC” ratio is another PK:PD parameter. It describes the maximumdrug concentration in plasma or tissue relative to the MIC.Fluoroquinolones and aminoglycosides are examples where C_(max):MIC maypredict in vivo bacterial killing where resistance can be suppressed.

“Time above MIC” (T>MIC) is another PK/PD parameter. It is expressed apercentage of a dosage interval in which the plasma or site-of-infectionlevel exceeds the MIC. Activity of antimicrobials with thetime-dependent killing (such as beta-lactams or monobactam antibioticss)is well predicted by the magnitude of the T>MIC ratio.

The term “dosing interval” refers to the time between administrations ofthe two sequential doses of a pharmaceutical's during multiple dosingregimens. For example, in the case of orally administered ciprofloxacin,which is administered twice daily (traditional regimen of 400 mg b.i.d)and orally administered levofloxacin, which is administered once a day(500 mg or 750 mg q.d.), the dosing intervals are 12 hours and 24 hours,respectively.

As used herein, the “peak period” of a pharmaceutical's in vivoconcentration is defined as that time of the pharmaceutical dosinginterval when the pharmaceutical concentration is not less than 50% ofits maximum plasma or site-of-infection concentration. In someembodiments, “peak period” is used to describe an interval ofantimicrobial dosing.

The estimated “respirable delivered dose” is the dose or amount of drugdelivered to the lung of a patient using a nebulizer or other aerosoldelivery device. The RDD is estimated from the inspiratory phase of abreath simulation device programmed to the European Standard pattern of15 breaths per minute, with an inspiration to expiration ratio of 1:1,and measurement of particles emitted from a nebulizer with a size ofabout 5 microns or less.

Improved Availability

The antibiotic rate of killing is dependent upon antibiotic mode ofaction and is determined by either the length of time necessary for theantibiotic to kill (time-dependent) or the effect of increasing theantibiotic concentration (concentration-dependent). Fluoroquinolones arecharacterized by concentration-dependent, time-kill activity where atherapeutic effect requires a high local peak concentration above theMICs of the infecting pathogen.

Fluoroquinolone efficacy in humans, animals and in vitro models ofinfection is linked to AUC:MIC ratio and C_(max):MIC ratio. A number ofin vitro studies have been conducted to determine if high concentrationsof levofloxacin with an extremely short half-lives (as predicted from arat and human PK model) in a target tissues resulted in bacterialkilling superior to that seen under conditions with more prolongedresidence times. In these studies, levofloxacin concentrations that were0.018-fold-1024-fold the MIC were evaluated in a standard kill-curve andan in vitro hollow fiber assay. In both of these assays, highconcentrations of levofloxacin were rapidly bactericidal and reachedtheir maximum levels of killing in 10-20 minutes. This level of killingwas sustained whether levofloxacin was maintained at that level or givena half-life of 10 minutes. In addition, no resistance was observed.Accordingly, high doses and rapid delivery of specially formulatedlevofloxacin is rapidly bactericidal for susceptible organisms andresistant organisms.

In one embodiment, the concentration of levofloxacin at the site ofinfection is increased by delivering levofloxacin in combination withdivalent or trivalent cations directly to the lung using inhalationtherapy, thereby decreasing the amount of time levofloxacin is in the“mutant selection window” (MSW). Such a therapeutic approach achievesbroader coverage of pathogens (including levofloxacin resistantstrains), prevents further resistance development, and results inshorter courses of levofloxacin therapy.

Some embodiments include compositions of levofloxacin or ofloxacinhaving an improved pulmonary availability, wherein an increasedpulmonary AUC is indicative of the improved pulmonary availability ofthe levofloxacin or ofloxacin. In some embodiments, the increase can beat least about 10%, 20, 30, 40%, 50%, 75%, 100%, 150%, 200%, 250%, 300%,and 500%. An increase can be relative to, for example, a compositionlacking a divalent or trivalent cation, and/or a composition havingcertain excipients (e.g., lactose), and/or a composition delivered tothe lung at a certain rate, and/or a certain respirable delivered dose.In some embodiments, methods are provided that include achieving animproved pulmonary availability indicated by a lung AUC greater thanabout 400 h·mg/L, about 500 h·mg/L, about 600 h·mg/L, about 700 h·mg/L,about 800 h·mg/L, about 900 h·mg/L, about 1000 h·mg/L, about 1100h·mg/L, about 1200 h·mg/L, about 1300 h·mg/L, about 1400 h·mg/L, about1500 h·mg/L, about 1600 h·mg/L, about 1700 h·mg/L, about 1800 h·mg/L,about 1900 h·mg/L, about 2000 h·mg/L, about 2100 h·mg/L, about 2200h·mg/L, about 2300 h·mg/L, about 2400 h·mg/L, about 2500 h·mg/L, about2600 h·mg/L, about 2700 h·mg/L, about 2800 h·mg/L, about 2900 h·mg/L,about 3000 h·mg/L, about 3100 h·mg/L, about 3200 h·mg/L, about 3300h·mg/L, about 3400 h·mg/L, about 3500 h·mg/L, about 3600 h·mg/L, about3700 h·mg/L, about 3800 h·mg/L, about 3900 h·mg/L, about 4000 h·mg/L,about 4100 h·mg/L, about 4200 h·mg/L, about 4300 h·mg/L, about 4400h·mg/L, and about 4500 h·mg/L. The increase can be measured for example,in bronchial fluid, homogenates of whole lung tissue, or in sputum.

In more embodiments, an increased pulmonary C_(max) can be indicative ofan improved pulmonary availability for a formulation of levofloxacin orofloxacin. In some such embodiments, the increase can be at least about50%, 75%, 100%, and 150%. An increase can be relative to a composition,for example, lacking a divalent or trivalent cation, and/or acomposition having certain excipients (e.g., lactose), and/or acomposition delivered to the lung at a certain rate, and/or a certainrespirable delivered dose. In some embodiments, methods are providedthat include achieving an improved pulmonary availability indicated by alung C_(max) greater than about 300 mg/L, about 400 mg/L, about 500mg/L, about 600 mg/L, about 700 mg/L, about 800 mg/L, about 900 mg/L,about 1000 mg/L, about 1100 mg/L, about 1200 mg/L, about 1300 mg/L,about 1400 mg/L, about 1500 mg/L, about 1600 mg/L, about 1700 mg/L,about 1800 mg/L, about 1900 mg/L, about 2000 mg/L, about 2100 mg/L,about 2200 mg/L, about 2300 mg/L, about 2400 mg/L, about 2500 mg/L,about 2600 mg/L, about 2700 mg/L, about 2800 mg/L, about 2900 mg/L,about 3000 mg/L, about 3100 mg/L, about 3200 mg/L, about 3300 mg/L,about 3400 mg/L, about 3500 mg/L, about 3600 mg/L, about 3700 mg/L,about 3800 mg/L, about 3900 mg/L, about 4000 mg/L, about 4100 mg/L,about 4200 mg/L, about 4300 mg/L, about 4400 mg/L, about 4500 mg/L,about 4600 mg/L, about 4700 mg/L, about 4800 mg/L, about 4900 mg/L, and5000 mg/L. The increase can be measured for example, in bronchialsecretions, epithelial lining fluid, lung homogenates, and in sputum.

In even more embodiments, a decrease in serum AUC or serum C_(max) canbe indicative of an increase in the pulmonary availability and prolongedexposure of a levofloxacin or ofloxacin using a formulation. In somesuch embodiments, the decrease can be at least about 1%, 5%, 10, 20%, or50%. A decrease can be relative to a composition, for example, lacking adivalent or trivalent cation, and/or a composition having certainexcipients (e.g., lactose), and/or a composition delivered to the lungat a certain rate as a solution or other composition. In someembodiments, a formulation of levofloxacin can be characterized by aAUC:MIC₉₀ greater than about 200, 300, 400, 500, 600, 700, 800, 900, and1000. In some such embodiments, the AUC can be pulmonary AUC.

In some embodiments, the concentrations in lung tissue (sputum, ELF,tissue homogenates) can be characterized by the PK-PD indiceC_(max):MIC₉₀ greater than about 20, 40, 60, 80, 100, 120, 140, 160,180, 200, 220, 240, 260, 280, 300, 320, 340, 340, 360, 380, 400, 420,440, 460, 480, 500, 520, 540, 560, 580, and 600.

The increase or decrease in a parameter to measure improved availabilityof a formulation of levofloxacin or ofloxacin can be relative to aformulation of levofloxacin or ofloxacin lacking divalent or trivalentcations, relative to a formulation of levofloxacin or ofloxacin lackinglactose, and/or relative to a formulation of with a lower concentrationof levofloxacin or ofloxacin.

Methods of Treatment or Prophylaxis

In some embodiments, a method is provided for treating a microbialinfection in an animal, specifically including in a mammal, by treatingan animal suffering from such an lung infection with a fluoroquinoloneantimicrobial formulated with a divalent or trivalent cation and havingimproved pulmonary availability. In some embodiments, fluoroquinoloneantimicrobials may be administered following aerosol formation andinhalation. Thus, this method of treatment is especially appropriate forthe treatment of pulmonary infections involving microbial strains thatare difficult to treat using an antimicrobial agent delivered orally orparenterally due to the need for high dose levels (which can causeundesirable side effects), or due to lack of any clinically effectiveantimicrobial agents. In one such embodiment, this method may be used toadminister a fluoroquinolone antimicrobial directly to the site ofinfection. Such a method may reduce systemic exposure and maximizes theamount of antimicrobial agent to the site of microbial infection. Thismethod is also appropriate for treating infections involving microbesthat are susceptible to fluoroquinolone antimicrobials as a way ofreducing the frequency of selection of resistant microbes. This methodis also appropriate for treating infections involving microbes that areotherwise resistant to fluoroquinolone antimicrobials as a way ofincreasing the amount of antimicrobial at the site of microbialinfection. A subject may be identified as infected with bacteria thatare capable of developing resistance by diagnosing the subject as havingsymptoms that are characteristic of a bacterial infection with abacteria species known to have resistant strains or a with a bacteriathat is a member of group that are known to have resistant strains.Alternatively, the bacteria may be cultured and identified as a speciesknown to have resistant strains or a bacteria that is a member of groupthat are known to have resistant strains.

In some embodiments, the aerosol fluoroquinolone antimicrobial agentformulated with divalent or trivalent cations is administered at a levelsufficient to overcome the emergence resistance in bacteria or increasekilling efficiency such that resistance does not have the opportunity todevelop.

In some embodiments, the aerosol fluoroquinolone therapy may beadministered as a treatment or prophylaxis in combination or alternatingtherapeutic sequence with other aerosol, oral or parenteral antibiotics.By non-limiting example this may include aerosol tobramycin and/or otheraminoglycoside, aerosol aztreonam and/or other beta or monobactam,aerosol ciprofloxacin and/or other fluoroquinolones, aerosolazithromycin and/or other macrolides or ketolides, tetracycline and/orother tetracyclines, quinupristin and/or other streptogramins, linezolidand/or other oxazolidinones, vancomycin and/or other glycopeptides, andchloramphenicol and/or other phenicols, and colisitin and/or otherpolymyxins.

In addition, compositions and methods provided herein can include theaerosol fluoroquinolone therapy administered as a treatment orprophylaxis in combination or alternating therapeutic sequence with anadditional active agent. As discussed above, some such additional agentscan include antibiotics. More additional agents can includebronchodilators, anticholinergics, glucocorticoids, eicosanoidinhibitors, and combinations thereof. Examples of bronchodilatorsinclude salbutamol, levosalbuterol, terbutaline, fenoterol, terbutlaine,pirbuterol, procaterol, bitolterol, rimiterol, carbuterol, tulobuterol,reproterol, salmeterol, formoterol, arformoterol, bambuterol,clenbuterol, indacterol, theophylline, roflumilast, cilomilast. Examplesof anticholinergics include ipratropium, and tiotropium. Examples ofglucocorticoids include prednisone, fluticasone, budesonide, mometasone,ciclesonide, and beclomethasone. Examples of eicosanoids includemontelukast, pranlukast, zafirlukast, zileuton, ramatroban, andseratrodast. More additional agents can include pulmozyme, hypertonicsaline, agents that restore chloride channel function in CF, inhaledbeta-agonists, inhaled antimuscarinic agents, inhaled corticosteroids,and inhaled or oral phosphodiesterase inhibitors. More additional agentscan include CFTR modulators, for example, VX-770, atluren, VX-809. Moreadditional agents can include agents to restore airway surface liquid,for example, denufosol, mannitol, GS-9411, and SPI-8811 More additionalagents can include anti-inflammatory agents, for example, ibuprofen,sildenafil, and simavastatin.

Pharmaceutical Compositions

For purposes of the method described herein, a fluoroquinoloneantimicrobial agent formulated with a divalent or trivalent cationhaving improved pulmonary availability may be administered using aninhaler. In some embodiments, a fluoroquinolone antimicrobial disclosedherein is produced as a pharmaceutical composition suitable for aerosolformation, good taste, storage stability, and patient safety andtolerability. In some embodiments, the isoform content of themanufactured fluoroquinolone may be optimized for tolerability,antimicrobial activity and stability.

Formulations can include a divalent or trivalent cation. The divalent ortrivalent cation can include, for example, magnesium, calcium, zinc,copper, aluminum, and iron. In some embodiments, the solution comprisesmagnesium chloride, magnesium sulfate, zinc chloride, or copperchloride. In some embodiments, the divalent or trivalent cationconcentration can be from about 25 mM to about 400 mM, from about 50 mMto about 400 mM, from about 100 mM to about 300 mM, from about 100 mM toabout 250 mM, from about 125 mM to about 250 mM, from about 150 mM toabout 250 mM, from about 175 mM to about 225 mM, from about 180 mM toabout 220 mM, and from about 190 mM to about 210 mM. In someembodiments, the concentration is about 200 mM. In some embodiments, themagnesium chloride, magnesium sulfate, zinc chloride, or copper chloridecan have a concentration from about 5% to about 25%, from about 10% toabout 20%, and from about 15% to about 20%. In some embodiments, theratio of fluoroquinolone to divalent or trivalent cation can be 1:1 to2:1 or 1:1 to 1:2.

Non-limiting fluoroquinolones for use as described herein includelevofloxacin, ofloxacin, ciprofloxacin, enoxacin, gatifloxacin,gemifloxacin, lomefloxacin, moxifloxacin, norfloxacin, pefloxacin,sparfloxacin, garenoxacin, sitafloxacin, and DX-619.

The formulation can have a fluoroquinolone concentration, for example,levofloxacin or ofloxacin, greater than about 50 mg/ml, about 60 mg/ml,about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190mg/ml, and about 200 mg/ml. In some embodiments, the formulation canhave a fluoroquinolone concentration, for example, levofloxacin orofloxacin, from about 50 mg/ml to about 200 mg/ml, from about 75 mg/mlto about 150 mg/ml, from about 80 mg/ml to about 125 mg/ml, from about80 mg/ml to about 120 mg/ml, from about 90 mg/ml to about 125 mg/ml,from about 90 mg/ml to about 120 mg/ml, and from about 90 mg/ml to about110 mg/ml. In some embodiments, the concentration is about 100 mg/ml.

The formulation can have an osmolality from about 300 mOsmol/kg to about500 mOsmol/kg, from about 325 mOsmol/kg to about 450 mOsmol/kg, fromabout 350 mOsmol/kg to about 425 mOsmol/kg, and from about 350 mOsmol/kgto about 400 mOsmol/kg. In some embodiments, the osmolality of theformulation is greater than about 300 mOsmol/kg, about 325 mOsmol/kg,about 350 mOsmol/kg, about 375 mOsmol/kg, about 400 mOsmol/kg, about 425mOsmol/kg, about 450 mOsmol/kg, about 475 mOsmol/kg, and about 500mOsmol/kg.

The formulation can have a pH from about 4.5 to about 8.5, from about5.0 to about 8.0, from about 5.0 to about 7.0, from about 5.0 to about6.5, from about 5.5 to about 6.5, and from 6.0 to about 6.5.

The formulation can comprise a conventional pharmaceutical carrier,excipient or the like (e.g., mannitol, lactose, starch, magnesiumstearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose,glucose, gelatin, sucrose, magnesium carbonate, and the like), orauxiliary substances such as wetting agents, emulsifying agents,solubilizing agents, pH buffering agents and the like (e.g., sodiumacetate, sodium citrate, cyclodextrine derivatives, sorbitanmonolaurate, triethanolamine acetate, triethanolamine oleate, and thelike). In some embodiments, the formulation can lack a conventionalpharmaceutical carrier, excipient or the like. Some embodiments includea formulation lacking lactose. Some embodiments comprise lactose at aconcentration less than about 10%, 5%, 1%, or 0.1%. In some embodiments,the formulation can consist essentially of levofloxacin or ofloxacin anda divalent or trivalent cation.

In some embodiments, a formulation can comprise a levofloxacinconcentration between about 75 mg/ml to about 150 mg/ml, a magnesiumchloride concentration between about 150 mM to about 250 mM, a pHbetween about 5 to about 7; an osmolality of between about 300 mOsmol/kgto about 600 mOsmol/kg, and lacks lactose.

In some embodiments, a formulation comprises a levofloxacinconcentration of about 100 mg/ml, a magnesium chloride concentration ofabout 200 mM, a pH of about 6.2, an osmolality of about 383 mOsmol/kg,and lacks lactose. In some embodiments, a formulation consistsessentially of a levofloxacin concentration of about 90 mg/ml to about110 mg/ml, a magnesium chloride concentration of about 180 mM to about220 mM, a pH of about 5 to about 7, an osmolality of about 300 mOsmol/kgto 500 mOsmol/kg, and lacks lactose.

Administration

The fluoroquinolone antimicrobials formulated with divalent or trivalentcations and having improved pulmonary availability may be administeredat a therapeutically effective dosage, e.g., a dosage sufficient toprovide treatment for the disease states previously described. Theamount of active compound administered will, of course, be dependent onthe subject and disease state being treated, the severity of theaffliction, the manner and schedule of administration and the judgmentof the prescribing physician; for example, a likely dose range foraerosol administration of levofloxacin would be about 20 to 300 mg perday, the active agents being selected for longer or shorter pulmonaryhalf-lives, respectively. In some embodiments, a likely dose range foraerosol administration of levofloxacin would be about 20 to 300 mg BID(twice daily).

Administration of the fluoroquinolone antimicrobial agents disclosedherein or the pharmaceutically acceptable salts thereof can be via anyof the accepted modes of administration for agents that serve similarutilities including, but not limited to, aerosol inhalation. Methods,devices and compositions for delivery are described in U.S. PatentApplication Publication No. 2006-0276483, incorporated by reference inits entirety.

Pharmaceutically acceptable compositions include solid, semi-solid,liquid and aerosol dosage forms, such as, for example, powders, liquids,suspensions, complexations, liposomes, particulates, or the like.Preferably, the compositions are provided in unit dosage forms suitablefor single administration of a precise dose.

The fluoroquinolone antimicrobial agent can be administered either aloneor in some alternatives, in combination with a conventionalpharmaceutical carrier, excipient or the like (e.g., mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodiumcrosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and thelike). If desired, the pharmaceutical composition can also contain minoramounts of nontoxic auxiliary substances such as wetting agents,emulsifying agents, solubilizing agents, pH buffering agents and thelike (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives,sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate,and the like). Generally, depending on the intended mode ofadministration, the pharmaceutical formulation will contain about 0.005%to 95%, preferably about 0.5% to 50% by weight of a compound of theinvention. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; for example, seeRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa.

In one preferred embodiment, the compositions will take the form of aunit dosage form such as vial containing a liquid, solid to besuspended, dry powder, lyophilate, or other composition and thus thecomposition may contain, along with the active ingredient, a diluentsuch as lactose, sucrose, dicalcium phosphate, or the like; a lubricantsuch as magnesium stearate or the like; and a binder such as starch, gumacacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivativesor the like.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier (e.g., water,saline, aqueous dextrose, glycerol, glycols, ethanol or the like) toform a solution or suspension. Solutions to be aerosolized can beprepared in conventional forms, either as liquid solutions orsuspensions, as emulsions, or in solid forms suitable for dissolution orsuspension in liquid prior to aerosol production and inhalation. Thepercentage of active compound contained in such aerosol compositions ishighly dependent on the specific nature thereof, as well as the activityof the compound and the needs of the subject. However, percentages ofactive ingredient of 0.01% to 90% in solution are employable, and willbe higher if the composition is a solid, which will be subsequentlydiluted to the above percentages. In some embodiments, the compositionwill comprise 1.0%-50.0% of the active agent in solution.

Compositions described herein can be administered with a frequency ofabout 1, 2, 3, 4, or more times daily, 1, 2, 3, 4, 5, 6, 7 or more timesweekly, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times monthly. Inparticular embodiments, the compositions are administered twice daily.

Aerosol Delivery

For pulmonary administration, the upper airways are avoided in favor ofthe middle and lower airways. Pulmonary drug delivery may beaccomplished by inhalation of an aerosol through the mouth and throat.Particles having a mass median aerodynamic diameter (MMAD) of greaterthan about 5 microns generally do not reach the lung; instead, they tendto impact the back of the throat and are swallowed and possibly orallyabsorbed. Particles having diameters of about 2 to about 5 microns aresmall enough to reach the upper- to mid-pulmonary region (conductingairways), but are too large to reach the alveoli. Smaller particles,i.e., about 0.5 to about 2 microns, are capable of reaching the alveolarregion. Particles having diameters smaller than about 0.5 microns canalso be deposited in the alveolar region by sedimentation, although verysmall particles may be exhaled.

In one embodiment, a nebulizer is selected on the basis of allowing theformation of an aerosol of a fluoroquinolone antimicrobial agentdisclosed herein having an MMAD predominantly between about 2 to about 5microns. In one embodiment, the delivered amount of fluoroquinoloneantimicrobial agent provides a therapeutic effect for respiratoryinfections. The nebulizer can deliver an aerosol comprising a massmedian aerodynamic diameter from about 2 microns to about 5 microns witha geometric standard deviation less than or equal to about 2.5 microns,a mass median aerodynamic diameter from about 2.5 microns to about 4.5microns with a geometric standard deviation less than or equal to about1.8 microns, and a mass median aerodynamic diameter from about 2.8microns to about 4.3 microns with a geometric standard deviation lessthan or equal to about 2 microns. In some embodiments, the aerosol canbe produced using a vibrating mesh nebulizer. An example of a vibratingmesh nebulizer includes the PARI E-FLOW® nebulizer or a nebulizer usingPARI eFlow technology. More examples of nebulizers are provided in U.S.Pat. Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911;4,510,929; 4,624,251; 5,164,740; 5,586,550; 5,758,637; 6,644,304;6,338,443; 5,906,202; 5,934,272; 5,960,792; 5,971,951; 6,070,575;6,192,876; 6,230,706; 6,349,719; 6,367,470; 6,543,442; 6,584,971;6,601,581; 4,263,907; 5,709,202; 5,823,179; 6,192,876; 6,644,304;5,549,102; 6,083,922; 6,161,536; 6,264,922; 6,557,549; and 6,612,303 allof which are hereby incorporated by reference in their entireties. Morecommercial examples of nebulizers that can be used with the formulationsdescribed herein include Respirgard II®, Aeroneb®, Aeroneb® Pro, andAeroneb® Go produced by Aerogen; AERx® and AERx Essence™ produced byAradigm; Porta-Neb®, Freeway Freedom™, Sidestream, Ventstream and I-nebproduced by Respironics, Inc.; and PARI LC-Plus®, PARI LC-Star®,produced by PARI, GmbH. By further non-limiting example, U.S. Pat. No.6,196,219, is hereby incorporated by reference in its entirety.

The amount of levofloxacin or ofloxacin that can be administered to thelungs with an aerosol dose, such as a respirable drug dose (RDD), thatcan include at least about 20 mg, about 30 mg, about 40 mg, about 50 mg,about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about110 mg, about 120 mg, about 125 mg, about 130 mg, about 140 mg, about150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about350 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, andabout 800 mg. In some embodiments, the amount of levofloxacin orofloxacin that can be administered to the lungs with an aerosol dose,such as a respirable drug dose (RDD), that can include at least about 20mg, 50 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg,500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg,950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg,1350 mg, 1400 mg, 1450 mg, and 1500 mg.

The aerosol can be administered to the lungs in less than about 10minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2minutes, and about 1 minute.

Indications

Methods and compositions described herein can be used to treat pulmonaryinfections and disorders. Examples of such disorders can include cysticfibrosis, pneumonia, and chronic obstructive pulmonary disease,including chronic bronchitis, and some asthmas. Some embodiments includetreating an infection comprising one or more bacteria selected from thegroup consisting of Pseudomonas aeruginosa, Pseudomonas fluorescens,Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida,Stenotrophomonas maltophilia, Aeromonas hydrophilia, Escherichia coli,Citrobacter freundii, Salmonella typhimurium, Salmonella typhi,Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae,Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacteraerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratiamarcescens, Morganella morganii, Proteus mirabilis, Proteus vulgaris,Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii,Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersiniaenterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersiniaintermedia, Bordetella pertussis, Bordetella parapertussis, Bordetellabronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae,Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilusducreyi, Pasteurella multocida, Pasteurella haemolytica, Helicobacterpylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli,Borrelia burgdorferi, Vibrio cholera, Vibrio parahaemolyticus,Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae,Neisseria meningitidis, Burkholderia cepacia, Francisella tularensis,Kingella, and Moraxella. In some embodiments, the lung infection iscaused by a gram-negative anaerobic bacteria. In more embodiments, thelung infection comprises one or more of the bacteria selected from thegroup consisting of Bacteroides fragilis, Bacteroides distasonis,Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroidesovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii, and Bacteroides splanchnicus. In some embodiments, the lunginfection is caused by a gram-positive bacteria. In some embodiments,the lung infection comprises one or more of the bacteria selected fromthe group consisting of Corynebacterium diphtherias, Corynebacteriumulcerans, Streptococcus pneumoniae, Streptococcus agalactiae,Streptococcus pyogenes, Streptococcus milleri; Streptococcus (Group G);Streptococcus (Group C/F); Enterococcus faecalis, Enterococcus faecium,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp.hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, andStaphylococcus saccharolyticus. In some embodiments, the lung infectionis caused by a gram-positive anaerobic bacteria. In some embodiments,the lung infection is caused by one or more bacteria selected from thegroup consisting of Clostridium difficile, Clostridium perfringens,Clostridium tetini, and Clostridium botulinum. In some embodiments, thelung infection is caused by an acid-fast bacteria. In some embodiments,the lung infection is caused by one or more bacteria selected from thegroup consisting of Mycobacterium tuberculosis, Mycobacterium avium,Mycobacterium intracellulare, and Mycobacterium leprae. In someembodiments, the lung infection is caused by an atypical bacteria. Insome embodiments, the lung infection is caused by one or more bacteriaselected from the group consisting of Chlamydia pneumoniae andMycoplasma pneumoniae.

EXAMPLES Comparative Example 1—Administration of Fluoroquinolones in aRat Pharmacokinetic Model

This example relates to aerosol and intravenous administration offluoroquinolones in saline. A rat pharmacokinetic model was used tocompare intravenous and pulmonary administration of fluoroquinolones.Male Sprague-Dawley rats (Charles Rivers) were administered 10 mg/kgdoses of levofloxacin, ciprofloxacin, gatifloxacin, norfloxacin, orgemifloxacin. Doses were administered via the lateral tail vein, or tothe lung just above the tracheal bifurcation using a micro-spray aerosoldevice (Penn Century, Philadelphia, Pa.). Levofloxacin was prepared insterile 0.9% saline to concentrations of 5 mg/ml (IV) and 60 mg/ml(aerosol).

Approximately 0.3 ml blood samples were taken from 2-6 rats at eachtimepoint via an indwelling jugular vein cannula, and collected inlithium heparin tubes. Bronchial alveolar lavage (BAL) and lung tissuewere collected following euthanasia. Levofloxacin concentrations inplasma, lung tissue and BAL were determined using a HPLC assay, and thedata analyzed using WinNonlin (Pharsight Corporation, v 5.0). Sampleconcentrations were determined against a standard curve.

Serum AUC_((0-inf)) (area under the concentration time curve, for timezero to infinity), serum MRT (mean retention time), serum t½(half-life), BAL AUC, MAT (mean absorption time), and F(bioavailability) were determined and are shown in Table 1.

TABLE 1 Serum AUC Serum Serum BAL MAT F, % from Drug Route (0-inf) MRTt½ AUC (h) Lung vs IV Levofloxacin IV 3.8 0.7 0.5 1.6 NA NA LevofloxacinAerosol 3.7 0.7 0.5 3.0 0 97% Ciprofloxacin IV 2.6 0.76 0.53 3.9 NA NACiprofloxacin Aerosol 0.8 1.35 0.93 78.4   0.59 82% Gatifloxacin IV 5.311.39 1.06 0.35 NA NA Gatifloxacin Aerosol 5.83 1.34 1.13 3.12 0 100% Norfloxacin IV 4.65 1.59 1.21 0.8 NA NA Norfloxacin Aerosol 4.46 1.291.13 24.6 0 100%  Gemifloxacin IV 4.54 1.41 1.04 0.9 NA NA GemifloxacinAerosol 5.86 2.06 1.68 140.4   0.65 86%

Aerosol administration of ciprofloxacin, gatifloxacin, norfloxacin, orgemifloxacin resulted in a significant increase in BAL AUC, compared tointravenous administration. Aerosol administration of levofloxacin didnot show such a significant increase in BAL AUC, compared to intravenousadministration. In addition, levofloxacin showed rapid absorption fromthe lung into serum. Thus, aerosol administration of levofloxacin insaline did not result in significant increased availability of drug tothe lung.

Comparative Example 2—Aerosol Administration of Levofloxacin withDivalent Cations in Rats

This example relates to a series of studies that included aerosoladministration of levofloxacin with divalent cations and lactose and IVor aerosol administration of levofloxacin in saline. Rats wereadministered 10 mg/kg levofloxacin (LVX) in saline or LVX formulatedwith CaCl₂, MgCl₂, or Zn⁺². Table 2 shows the formulations oflevofloxacin used in these studies.

TABLE 2 Levo- Levo- Levo- Levo- Levo- floxacin floxacin floxacinfloxacin floxacin (IV) (Aerosol) (MgCl₂) (CaCl₂) (ZnCl₂) Levo- 5 mg/ml60 mg/ml    60 mg/ml    60 mg/ml    60 mg/ml floxacin MgCl₂ — — 120 mM —— CaCl₂ — — — 120 mM — ZnCl₂ — — — — 120 mM Lactose — — 150 mM — —

In one study, pharmacokinetic parameters including C_(max) (maximumserum concentration), CL/F (total body clearance/bioavailability) weremeasured and are shown in Table 3. A graph of plasma concentration oflevofloxacin with time is shown in FIG. 1, where levofloxacin wasadministered by aerosol, by intravenous injection, or by aerosol withMgCl₂.

TABLE 3 Mean (+/− SD) Parameter Unit LVX IV LVX Aerosol LVX MgCl₂ PlasmaAUC hr · mg/L 3.79 (±0.89) 3.69 (±0.14) 3.72 (±0.24) Plasma Half-life hr0.49 (±0.10) 0.52 (±0.09) 0.73 (±0.07) Plasma C_(max) mg/L 5.54 (±1.51)6.01 (±1.54) 6.66 (±1.70) Plasma CL/F L/hr/kg 2.81 (±0.54) 2.83 (±0.11)2.68 (±0.17) Plasma MRT hr 0.70 (±0.14) 0.71 (±0.08) 0.88 (±0.06) PlasmaMAT hr NA 0.01 0.18 Plasma F % NA 97.4 98.2 (Bioavailability) BALAUC_((0-6 h)) hr · mg/L 1.6 3.0 8.3

A two compartment pharmacokinetic model may be used describe thedifference in graphs of plasma levofloxacin with time for intravenousand aerosol administration. Plasma AUC after intravenous administrationwas similar to plasma AUC after administration by aerosol with Mg⁺²(3.79 hr·mg/L vs. 3.72 hr·mg/L, respectively). This suggests near 100%bioavailability of the divalent-complex antibiotic from the lung. Themean residence time (MRT) of levofloxacin was greater after aerosoladministration compared to after intravenous administration (0.88 vs.0.70 hours). This delay in absorption was associated with an increase inBAL levofloxacin AUC_((0-6h)) in BAL (1.6 hr·mg/L vs. 8.3 hr·mg/L forintravenous vs. aerosol dosing, respectively), and an 18-fold increasein the mean absorption time (MAT)

In another study, levofloxacin levels after aerosol administration forformulations containing saline, Zn⁺², Ca⁺² or Mg⁺² were measured andpharmacokinetic parameters were determined. Table 4 and FIG. 2 summarizethe results.

TABLE 4 Serum AUC Serum Serum BAL MAT F, % from Drug Route (0-inf) MRTt½ AUC (h) Lung vs IV Levofloxacin IV 3.8 0.7 0.5 1.6 NA NA LevofloxacinAerosol 3.7 0.7 0.5 3.0 0  97% Levofloxacin Aerosol 4.4 1.35 1.2 29.60.7 116% (MgCl₂) Levofloxacin Aerosol 4.3 1.17 0.8 8.3 0.5 116% (CaCl₂)Levofloxacin Aerosol 4.4 1.6 1.8 55.6 0.9 100% (ZnCl₂)

Aerosol administration of levofloxacin complexed with Ca′ and Mg′resulted in a longer plasma half-life and longer MAT compared tolevofloxacin formulated in saline, indicative of slower lung clearanceto plasma (Table 4). Levofloxacin formulated with Ca′ or Mg′ produced a2- to 5-fold higher levofloxacin C_(max) and AUC in BAL and lung tissuecompared to intravenous levofloxacin or aerosolized levofloxacinformulated in saline (Table 4, FIG. 2). These data suggest that aerosollevofloxacin complexed with divalent cation should result in higherefficacy in the treatment of pulmonary infections.

Example 3—Pharmacokinetic Modeling and Deconvolution Analysis

This example relates to modeling drug concentrations in lung.Pharmacokinetic deconvolution methods are useful to determine the amountof drug remaining in the lung after administration. Such methods areparticularly useful where direct measurements are difficult and/orproduce variable results, for example, measuring drug concentrations inlung using sputum samples.

Serum and urinary pharmacokinetic parameters can be determined usingnon-compartmental and compartmental methods, and drug concentrations inthe lung over time can be calculated using deconvolution. This approachhas been reported for aerosol delivery of tobramycin, where a dose of5.6 mg/kg showed bioavailability of about 9% and absorption over a 3hour period, consistent with empirically derived data (Cooney G. F., etal, “Absolute bioavailability and absorption characteristics ofaerosolized tobramycin in adults with cystic fibrosis.” J. ClinicalPharmacol. (1994), 34, 255-259, incorporated herein by reference in itsentirety).

An example deconvolution method is summarized in FIG. 3. This analysiscompares the appearance and elimination of drug following aerosol andintravenous doses to determine the amount of drug remaining in the lung(absorption compartment) over time. To estimate concentrations of drugin lung, amounts were divided by estimates of the epithelial lung fluid(ELF) volume (25 ml) for each subject. Non-compartmental pharmacokineticanalysis was subsequently applied to these projected concentrations ofdrug in the lung to determine AUCs.

Application of the deconvolution methodology can be accomplished byhuman or animal aerosol dosing of 50 mg respirable drug dose oflevofloxacin in saline or complexed with Mg⁺², delivered in a nebulizeror other respiratory delivery device, with resulting plasma drugconcentrations profiles and calculated pharmacokinetic parameters asillustrated in FIG. 4 and Tables 5 and 6. Serum levofloxacinconcentrations following a 5 minute IV infusion of levofloxacin to asingle healthy volunteer were analyzed using WinNonlin and thepharmacokinetic parameters presented in Table 5. On a separate occasion,this volunteer received a single aerosol dose of levofloxacin (RDD=50mg) by a PARI eFlow vibrating mesh nebulizer. FIG. 4 shows a comparisonof the serum levofloxacin concentrations following an IV or aerosoldose. Using the PK model depicted in FIG. 3, serum concentrations oflevofloxacin measured in serum following an aerosol dose weredeconvoluted using the serum PK data for an IV dose of levofloxacin (PKparameters shown in Table 5). The results are shown in Table 6, whichshow the estimated amount of levofloxacin (in mg) remaining in the lungover time.

TABLE 5 Parameter Unit Estimate AUC hr · mg/L 3.14 K10_HL hr 0.92 Alpha1/hr 6.85 Beta 1/hr 0.11 Alpha_HL hr 0.10 Beta_HL hr 6.50 A mg/L 2.05 Bmg/L 0.30 C_(max) mg/L 1.88 CL L/hr 15.93 AUMC hr · hr · mg/L 26.78 MRThr 8.49 Vss L 135.29 V2 L 114.03 CLD2 L/hr 111.26

TABLE 6 Cumulative Drug Input rate input into Input remaining FractionHours (mg/hr) compartment (mg) fraction in lung (mg) remaining 0.2435.79 26.67 0.53 23.33 0.47 0.48 13.80 31.16 0.62 18.84 0.38 0.72 18.2534.84 0.70 15.16 0.30 0.96 20.55 39.88 0.80 10.12 0.20 1.2 14.98 44.140.88 5.86 0.12 1.44 9.21 47.03 0.94 2.97 0.06 1.68 5.13 48.68 0.97 1.320.03 1.92 1.81 49.51 0.99 0.49 0.01 2.16 0.45 49.69 0.99 0.31 0.01 2.40.36 49.79 1.00 0.21 0.00 2.64 0.27 49.86 1.00 0.14 0.00 2.88 0.18 49.921.00 0.08 0.00 3.12 0.11 49.95 1.00 0.05 0.00 3.36 0.08 49.97 1.00 0.030.00 3.6 0.05 49.99 1.00 0.01 0.00 3.84 0.02 50.00 1.00 0.00 0.00

These data can be used to calculate the amount (in mg) of levofloxacinremaining in the lung as a function of time. FIG. 5 and Table 6 shows anexample for the estimated amount of drug remaining in the lung overtime, whereas only 10% of the 50 mg respirable drug dose administeredover a 5 minute period remains in the lung after 1.2 hours. Thisexperiment demonstrates the utility of the deconvolution method.

Comparative Example 4—Aerosol and Systemic Administration ofLevofloxacin with Saline

This example relates to aerosol and systemic administration oflevofloxacin formulated in a saline solution using estimated respirabledrug doses of 20 mg or 40 mg (nebulizer loaded doses of 43.3 and 86.6mg, respectively) of levofloxacin. Single aerosol doses of two doselevels levofloxacin (using the IV formulation Levaquin®) wereadministered to normal healthy volunteers and stable CF subjects usingthe PARI eFlow high efficiency nebulizer.

Safety, tolerability, and pharmacokinetics (serum, sputum, and urinaryexcretion) data were collected after each dose. The nebulizer was loadedwith 3.6 ml of a Levaquin® solution diluted in saline to isotonicity, ata concentration of 11.9 mg/ml for the 20 mg respirable drug dose group,and at a concentration of 23.8 mg/ml for the 40 mg respirable drug dosegroup. These volumes correspond to “load” doses of 43.3 mg and 86.6 mglevofloxacin for the 20 and 40 mg RDD, respectively. Table 7 summarizesnebulizer loaded doses with the corresponding estimated RDD forlevofloxacin formulated in saline.

TABLE 7 Levofloxacin Nebulizer RDD (based Concentration Loaded dose onparticles < in Saline (mg/L) (mg) 5 μm) (mg) 12 43.3 20 23.8 86.6 40

Novaluzid® (AstraZeneca) was co-administered to minimize the oralabsorption of any levofloxacin that was swallowed during inhalation.Each subject received an intravenous dose of levofloxacin and an aerosolsaline dose at the first visit to generate pharmacokinetic data forcomparison with aerosol levofloxacin doses, and to assess thetolerability of delivering solutions using the eFlow device.

Serum and urine levofloxacin concentrations were analyzed using avalidated HPLC assay by Anapharm (Quebec City, Canada). Sputumlevofloxacin assays were developed and cross validated using the serumassay.

Serum data: Serum levofloxacin concentrations following the intravenousinfusion were fit to a two-compartment open pharmacokinetic model usingiteratively reweighted least-squares regression (WinNonlin). A weight of1/y-observed was applied in the regression. Goodness of fit was assessedby the minimized objective function and inspection of weighted residualplots. Serum levofloxacin concentrations resulting from aerosoladministration were analyzed using deconvolution methods to estimate theresidence time of the aerosol dose in the lung (Gibaldi M. and PerrierD. Pharmacokinetics, 2nd Edition. Marcel Dekker: New York, 1982,incorporated herein by reference in its entirety). The pharmacokineticmodel and approach used for deconvolution analysis is described inExample 3. Briefly, this analysis compares the appearance andelimination of drug following aerosol and intravenous doses to determinethe amount of drug remaining in the lung (absorption compartment) overtime. To estimate concentrations of drug in lung, amounts were dividedby estimates of the epithelial lung fluid (ELF) volume (25 ml) for eachsubject. Noncompartmental pharmacokinetic analysis was subsequentlyapplied to these projected concentrations of drug in the lung todetermine values for AUC.

Sputum data: Sputum concentration data were analyzed usingnoncompartmental pharmacokinetic methods (Gibaldi M. and Perrier D.Pharmacokinetics, 2^(nd) Edition. Marcel Dekker: New York, 1982,incorporated herein by reference in its entirety). The area under thesputum concentration vs. time curve was estimated using the lineartrapezoidal rule. Since sputum was only collected from 0.5 to 8 hrs,forward and backward extrapolation from terminal and initial phases wasconducted to generate estimates of secondary pharmacokinetic parameters(C_(max), AUC).

PK-PD parameters such as AUC:MIC, and C_(max):MIC, were generated forlung exposures estimated from deconvolution of serum levofloxacinconcentration data. Examples of parameters were calculated at differentvalues for levofloxacin MIC for P. aeruginosa at estimated respirabledoses of levofloxacin ranging from 20-120 mg administered twice daily inCF subjects. Levofloxacin MIC distributions (MIC₅₀, MIC₉₀, and mode MIC)were measured for clinical isolates from CF isolates (Traczewski MM andBrown SD. In Vitro activity of doripenem against P. aeruginosa andBurkholderia cepacia isolates from both cystic fibrosis and non-cysticfibrosis patients. Antimicrob Agents Chemother 2006; 50:819-21,incorporated herein by reference in its entirety).

Dosing summary: A total of 7 normal healthy volunteers (NHV) and 9subjects with CF were enrolled in the study. All subjects completed allphases of the protocol; the dose summary is provided in Table 8. Sevenof 9 cystic fibrosis subjects received both the 20 and 40 mg respirabledrug dose levels, whereas 2 subjects were re-dosed with the 20 mg doselevel with salbutamol pretreatment. Forced expiratory volume during 1second (FEV₁)

TABLE 8 Baseline FEV₁ Levofloxacin Doses Studied Subject Gender (% Pred)20 mg 40 mg 20 mg A* Normal healthy volunteers 001 M  83% X X 002 M 100%X X 003 M 109% X X 004 M 130% X X 006 M 119% X X 008 M  82% X X 009 F104% X X Cystic fibrosis patients 010 F  58% X X 012 M  67% X X 013 F 40% X X 014 F  40% X X 015 M  74% X X 016 F  66% X X 018 M  63% X X 019M  42% X X 022 M  48% X X *A = Repeat study using pretreatment withsalbutamol

Levofloxacin pharmacokinetics in serum: FIG. 6 shows mean serumlevofloxacin concentrations in normal and CF subjects following IV andaerosol doses. Total levofloxacin clearance was 17.2 and 14.1 L/h in theNHV and CF subjects, respectively. Serum levofloxacin concentrationsfollowing aerosol administration generally paralleled those observedwith the intravenous dose, particularly after 1 hour post-dosing.Comparison of the levofloxacin AUCs from IV or aerosol dosing usingmodel-independent analysis showed that levofloxacin exposure fromaerosol doses relative to the 50 mg IV dose was (mean+/−SD) 35.6+/−9.4%and 59.4+/−16.6% for the low and high aerosol doses, respectively forthe normal volunteers, and 27.9+/−3.3% and 51.1+/−11.2% for CF subjects.

Serum deconvolution analysis: Serum levofloxacin concentrationsfollowing aerosol administration were successfully deconvoluted in allsubjects, permitting estimation of the amount of drug in the absorption(lung) compartment over time (FIG. 7). Absorption from the lung intoserum occurred significantly more slowly in CF subjects than in healthynormal volunteers; 50% of the lung dose appeared to remain in the lungfor at least 0.5 hours after the dose. Using a literature value forestimation of lung epithelial lining fluid (ELF) volume of 25 ml, theestimated concentrations of levofloxacin in ELF of CF patients followinga single aerosol dose is shown in FIGS. 8A and 8B (Rennard, S, G. etal., Estimation of volume of epithelial lining fluid recovered by lavageusing urea as a marker of dilution. J. Appl. Physio. 60: 532-8,incorporated by reference herein in its entirety). Mean projectedC_(max) concentrations in the ELF of CF patients at the conclusion ofthe low and high doses exceeded 500 μg/ml and 1000 μg/ml, respectively.When integrated over time, the projected mean+/−SD levofloxacin AUC inlung fluid was 365+/−338 and 710+/−471 for the low and high dose inhealthy subjects, and 354+/−274 and 1,199+/−1,147 for low and high dosesin CF patients.

Levofloxacin pharmacokinetics in sputum: FIG. 9 shows levofloxacinconcentrations in sputum following administration of the low and highaerosol dose in CF subjects. Sputum levofloxacin concentrationsfollowing both aerosol dose levels were markedly higher for at least 1hour post-dose with aerosol levofloxacin than those obtained with the 50mg IV dose. Concentrations tended to fall rapidly during the first 2hours of administration, consistent with drug absorption from the lung.Sputum levofloxacin concentrations were variable within and betweenpatients, but generally the 86.6 mg loaded dose did provide higherconcentrations over the period of observations.

Levofloxacin concentrations in sputum from CF subjects following aerosoladministration were averaged and compared with concentrations obtainedby other routes of administration. FIG. 10 depicts modeled sputumconcentrations in CF subjects for both aerosol dose levels, the 50 mg IVdose to the same subjects infused over 5 minutes, and a 750 mg oral dose(another study not described herein); Table 9 shows C_(max),AUC, andhalf-life values for measured sputum levofloxacin concentrations.

TABLE 9 50 mg 20 mg 40 mg 750 mg Parameter IV Aerosol Aerosol OralC_(max) (mg/L) 0.8 86.2 211.5 8.7 AUC (hr · mg/L) 2.5 67.1 171.4 93.4T_(1/2) (h) 3.8 0.9 1.3 6.7

While a 750 mg oral levofloxacin dose results in more prolonged drugconcentrations in sputum, aerosol doses as low as 20 mg produce peakconcentrations 10-fold higher.

FIG. 6 shows serum levofloxacin concentrations following single RDDs of20 or 40 mg of levofloxacin formulated in saline, and IV doses ofLevaquin to normal healthy volunteers and CF patients. These data wereused to perform pharmacokinetic deconvolution as previously described.FIG. 7 shows the results of deconvolution, showing the estimated amountof levofloxacin remaining in the lung over time. Levofloxacin remainedin the lung for a longer period in CF patients compared to normalhealthy volunteers (FIG. 7). Notably, the levofloxacin concentrationsobserved in sputum are consistent with ELF concentrations projected fromthe deconvolution analysis (FIGS. 8A and 8B vs. FIG. 9 and Table 9).

PK-PD analysis: Integration of pharmacokinetics with susceptibility datafor P. aeruginosa allows for assessment of the expected pharmacodynamiceffects in vivo. PK-PD parameters for fluoroquinolones include the 24 hrAUC:MIC and C_(max):MIC ratios. Very high C_(max):MIC ratios appear tobe significant for rapid bacterial killing and suppression of drugresistance.

The results of PK-PD analysis with simulated ELF PK data (generated fromthe amount of levofloxacin in lung divided by the ELF volume) from thedeconvolution analysis for twice daily dosing of levofloxacin along withMIC data for P. aeruginosa can be used to calculate levofloxacin PK-PDindices for P. aeruginosa. Table 10 shows predicted PK-PD indices(C_(max):MIC; 24 h AUC:MIC) for particular dosage regimens oflevofloxacin.

TABLE 10 Levofloxacin C_(max):MIC (24 h AUC:MIC) values for MIC*Levofloxacin dosage regimens (mg/L) 20 mg BID 40 mg BID 80 mg BID 120 mgBID 32 16 (22)   31 (44) 62 (88)  94 (131) 16 (MIC₉₀) 31 (44)   62 (88)124 (175) 188 (263) 8 62 (88)   124 (175) 248 (350) 375 (525) 4 124(175)   248 (350) 496 (700)   750 (1,050) 2 248 (350)   496 (700)   992(1,400) 1,500 (2,100) 1 (MIC₅₀) 496 (700)     992 (1,400) 1,884 (2,800)3,000 (4,200) 0.5 (mode) 992 (1,400) 1,884 (2,800) 3,768 (4,200) 6,000(8,400) 24 h AUC 700 1,400 2,800 4,200 (h · mg/L) C_(max) 500 1,0002,000 3,000 (mg/L) *MIC₅₀, MIC₉₀, and mode values from Traczewski M Mand Brown S D (2006)

For example, a daily dose of 20 mg BID levofloxacin, C_(max):MIC=248; 24h AUC:MIC=350; and a levofloxacin MIC=2 mg/L. The simulations show thatthe primary target value of C_(max):MIC >20 would be obtained by allregimens for over 90% of CF isolates of P. aeruginosa. In addition, thesecondary PK-PD target value of 24 hr AUC:MIC>300 would be obtained fora majority of strains at the lower doses, but could also cover over 90%of the isolates at the higher doses projected to be evaluated inupcoming clinical studies.

Comparative Example 5—Aerosol Administration of 30 mg/ml and 50 mg/mlSolutions of Levofloxacin Formulated with MgCl₂

This example relates to aerosol administration to CF patients of 30mg/ml and 50 mg/ml solutions of levofloxacin formulated with MgCl₂.Table 11 shows the formulations of levofloxacin with MgCl₂ and lactose.

TABLE 11 30 mg/ml 50 mg/ml Levofloxacin, mg/ml (mM)    30 (81.6)   50(136) Magnesium, mg/ml (mM) 1.5 (60)  2.4 (100) Chloride, mg/ml (mM) 4.3 (120)  7.1 (200) Lactose, mg/ml (mM) 51.4 (150) 51.4 (150) pH 6.36.3 Osmolality, mOsm/kg 314 400

Eight stable CF patients received loaded doses of 78 mg, 175 mg, and 260mg (corresponding to RDD of 40 mg, 80 mg, and 120 mg, respectively) oflevofloxacin formulated with MgCl₂ using an eFlow high efficiencynebulizer (PART Pharma, Munich, Germany). Escalated doses wereadministered 1 week apart. A separate group of 7 CF patients wereadministered a single dose of 750 mg oral levofloxacin at weeklyintervals for 4 consecutive weeks. Serum and sputum samples were assayedfor levofloxacin by HPLC. Serum and sputum levofloxacin concentrationdata were analyzed using non-compartmental pharmacokinetic methods. Meanpharmacokinetic parameters are shown in Table 12.

TABLE 12 Dose Aerosol Aerosol Aerosol 78 mg 175 mg 260 mg Oral IVParameter RDD: 40 mg RDD: 80 mg RDD: 120 mg 750 mg 50 mg Sputum C_(max)388 714 1112 8.7 1.05 (mg/L) C_(max): MIC₉₀* 49 89 139 1.1 0.1AUC_((0-inf)) 851 656 1448 93.4 5.70 (h · mg/L) t½ (h) 3.09 1.61 2.516.70 3.5 Serum C_(max) 0.48 0.95 1.30 7.30 2.55 (mg/L) AUC_((0-inf))2.08 4.45 6.54 76.6 3.91 (h · mg/L) t½ (h) 5.69 6.50 6.20 7.60 5.89MAT** 1.06 1.61 1.30 ND ND *P. aeruginosa MIC₉₀ for CF isolates is 8μg/ml MAT = Mean absorption time form the lung.

PK-PD data have previously shown that for fluoroquinolones, C_(max):MICratio is a PK-PD parameter associated with optimal bacterial killing andprevention of resistance. Aerosol administration of levofloxacin withMgCl₂ provides concentrations in sputum that achieve C_(max):MIC ratiosfor P. aeruginosa >40. In contrast, an oral levofloxacin dose of 750 mgproduces a ratio of 1.1. These data show that aerosolized doses oflevofloxacin with MgCl₂ provide high exposures in sputum that aregreater than those achievable with oral levofloxacin.

Comparative Example 6—Comparison of Aerosol Administration of a 40 mgRDD of Levofloxacin Formulated in Saline or MgCl₂ in CF Patients

This example relates to aerosol administration of levofloxacin withMgCl₂ or in saline using estimated respirable drug doses (RDD) of 40 mglevofloxacin. The concentrations of levofloxacin in saline are 23.8mg/ml and 30 mg/ml in a formulation containing MgCl₂/lactose (see Table11). CF patients received 40 mg respirable drug doses of levofloxacin byaerosol delivery: 7 patients received levofloxacin formulated in saline;10 patients received the same estimated RDD received levofloxacinformulated with MgCl₂. Sputum samples were taken at various times up to24 hours and levofloxacin concentrations determined using aHPLC/fluorescence method. Mean levofloxacin concentrations measured insputum over time are shown in FIG. 11. Levofloxacin delivered with MgCl₂is retained in sputum for a longer period and at higher concentrationsthan the same dose of levofloxacin delivered in saline.

Further comparison of the PK parameters in CF sputum for aerosoladministration of levofloxacin in saline (Example 4-Table 8) indicatethat both a significantly higher sputum C_(max) and AUC are achieved bycomplexation with magnesium (e.g., C_(max) is 211.5 mg/L levofloxacinvs. 388 for levofloxacin:Mg and AUC is 171.4 h·mg/L levofloxacin/salinevs. 851 h·mg/L levofloxacin:Mg for 40 mg respirable dose).

Example 7: Pharmacokinetics of Levofloxacin in CF Patients FollowingAerosol Administration of Formulations Containing MgCl₂ Plus Lactose forUp to 14 Days

CF patients received respirable delivered doses of approximately 40 mg,80 mg, or 120 mg per treatment (loaded doses of 78 mg, 175 mg, or 260 mgper treatment) on day 1 followed by twice daily dosing for 14 days.Formulations shown in Table 11 were used. Standard non-compartmental andcompartmental PK methods were used to generate serum, sputum, andurinary PK parameters (Gibaldi M, Perrier B. Pharmacokinetics. 2nd ed.New York:Marcel-Dekker; 1982, incorporated by reference herein in itsentirety). PK parameters were determined for serum and sputum and areshown in Tables 13 and 14, respectively. Comparison with theadministration of levofloxacin in saline (Example 4) indicate that botha significantly higher sputum C_(max) and AUC are achieved bycomplexation with magnesium (e.g., C_(max) is 211.5 mg/L levofloxacinvs. 448.97 for levofloxacin:Mg and AUC is 171.4 h·mg/L levofloxacin vs.420.54 h·mg/L levofloxacin:Mg (day 1) for 40 mg respirable dose).

TABLE 13 Loaded Levofloxacin Dose (Mean ± SD) 78 mg 175 mg 260 mg (n =10) (n = 10) (n = 10) Parameter RDD: 40 mg RDD: 80 mg RDD: 120 mg Day 1Serum C_(max) (mg/L) 0.36 ± 0.6  1.05 ± 0.29 1.34 ± 0.42 Serum T_(max)(h) 0.21 1.00 0.29 Serum AUC_((0-t)) 2.2.61 ± 9.12 ± 1.89 10.24 ± 3.08 (h · mg/L) 1.81 Serum AUC_((inf)) 3.40 ± 1.69 9.94 ± 2.30 11.44 ± 2.86 (h · mg/L) Serum t½ (h) 7.21 ± 1.89 6.60 ± 0.91 7.36 ± 2.42 Day 15 SerumC_(max) (mg/L) 0.58 ± 0.35 1.37 ± 0.56 2.39 ± 0.56 Serum T_(max) (h)0.53 0.98 0.22 Serum AUC_((0-t)) 5.16 ± 1.22 12.95 ± 5.75  18.12 ± 12.00(h · mg/L) Serum t½ (h) 8.48 ± 2.42 6.52 ± 0.87 6.62 ± 1.16

TABLE 14 Loaded Levofloxacin Dose (Mean ± SD) 78 mg (n = 10) 175 mg (n =10) 260 mg (n = 10) Parameter RDD: 40 mg RDD: 80 mg RDD: 120 mg Day 1Sputum C_(max) (mg/L) 448.97 ± 875.02  1333.96 ± 1146.55 1766.23 ±1493.52 Sputum T_(max) (h) 0.52 0.53 0.54 Sputum AUC_((0-t)) (h · mg/L)420.54 ± 994.99  1468.60 ± 1420.04 1779.23 ± 1223.12 Sputum t½ (h) 1.54± 0.56 2.56 ± 1.94 5.04 Day 15 Sputum C_(max) (mg/L) 612.06 ± 1440.131258.82 ± 1888.15 1721.51 ± 1511.15 Sputum T_(max) (h) 0.52 0.53 0.50Sputum AUC_((0-t)) (h · mg/L) 637.56 ± 1280.39 1642.81 ± 2849.76 1272.76± 795.19  Sputum t½ (h) 9.96 ± 13.9  4.10 ± 1.93 2.73 ± 1.58

Example 8—Aerosol Administration of 50 mg/ml and 100 mg/ml Solutions ofLevofloxacin Formulated with MgCl₂

This example relates to aerosol administration to CF patients of 50mg/ml and 100 mg/ml solutions of levofloxacin formulated with MgCl₂ atdoses of 180 mg and 240 mg. Table 15 shows the formulations oflevofloxacin with MgCl₂.

TABLE 15 50 mg/ml 100 mg/ml Levofloxacin, mg/ml 50 100 Magnesium, mg/ml(mM)  2.4 (100)  4.9 (200) Chloride, mg/ml (mM)  7.1 (200) 14.2 (400)Lactose, mg/ml (mM) 51.4 (150)  0 (0) pH 6-8 6-8 Osmolality, mOsm/kg300-500 300-500

Levofloxacin with MgCl₂ was administered by inhalation using a PARIeFlow nebulizer using vibrating mesh technology with the 35 L headconfiguration. Subjects received, in an order specified by arandomization schedule, a single 180 mg dose of a particular formulation(50 mg/ml or 100 mg/ml) in Period 1 of the study, followed by a 7-daywash-out period and a single 180 mg dose of the other formulation (50mg/ml or 100 mg/ml) in Period 2. This was followed by 7 consecutive daysof a once-daily 240 mg dose during Period 3. Serum and sputumconcentrations of levofloxacin were measured using an HPLC/fluorescencemethod.

With respect to serum concentrations of levofloxacin, the arithmeticmean serum concentrations of levofloxacin after administration of 180 mgwith the 100 mg/ml formulation were slightly higher than afteradministration with the 50 mg/ml formulation (FIG. 12). Table 16summarizes pharmacokinetic parameters for levofloxacin afteradministration of single 180 mg doses as a 50 mg/ml or 100 mg/mlsolution for inhalation, and after administration of 240 mg as a 100mg/ml solution for inhalation once daily for 7 days to patients with CF.The mean C_(max) and AUC_((inf)) for the 100 mg/ml formulation were 35%and 22% higher than the corresponding values for the 50 mg/mlformulation.

TABLE 16 Dose: 180 mg Dose: 240 mg Parameter¹ 50 mg/ml 100 mg/ml 100mg/ml Serum C_(max) (ng/ml) 952 ± 617 1,284 ± 1,707 ± 624 (10)   642(10) (10) Serum T_(max) (h)  0.25 (10)  0.17 (10)  0.3 (10) SerumC_(min) (ng/ml) 58.5 ± 60.4 73.5 ± 45.8   145 ± 172 (10) (10) (10) SerumAUC_((0-t))(h · ng/ml) 7,074 ± 9,054 ± 14,771 ± 3,625 (10) 3,411 (10)9,969 (10) Serum AUC_((inf)) (h · ng/ml) 8,058 ± 9,848 ± 16,930 ± 3,704(9)  3,813 (10) 13,631 (10)  Serum t½ (h) 6.40 ± 1.27 6.78 ± 1.61   7.49± 2.89  (9) (10) (10) ¹Arithmetic mean ± standard deviation (N) exceptfor T_(max) for which the median (N) is reported.

Based on a mean t½ of 6.78 h after administration of 180 mg with the 100mg/ml formulation, the accumulation with once-daily dosing should beabout 9%. There was a 1.33-fold increase in the mean C_(max) afteradministration of 240 mg with the 100 mg/ml formulation, similar to theincrease in level of dose. AUC_((0-t)) on Day 7 after administration of240 mg QD×7 days is AUC₍₀₋₂₄₎, or the AUC over the dosing interval,which should be equivalent to AUC_((inf)) after a single dose.Correcting the 14,771 h·ng/ml mean AUC_((0-t)) of the 240 mg dose levelto the 180 mg dose level, results in an estimate of 11,078 h·ng/ml,comparable to the observed AUC_((inf)) of 9,848±3,813 h·ng/ml afteradministration of a single 180 mg dose of the same formulation. Thisdemonstrates the linearity of the pharmacokinetics of levofloxacin aftersingle and multiple aerosol doses of levofloxacin with the 100 mg/mlformulation. The arithmetic mean t½ was comparable for all threetreatments, ranging from 6.40 h to 7.49 h.

With respect to sputum concentrations of levofloxacin, mean values forarithmetic sputum concentration, C_(max), and AUC were similar afteradministration of 180 mg with either the 50 mg/ml or 100 mg/mlformulation (FIG. 13). Table 17 summarizes sputum pharmacokineticparameters for levofloxacin after administration of single 180 mg dosesas a 50 mg/ml or 100 mg/ml solution for inhalation, and afteradministration of 240 mg as a 100 mg/ml solution for inhalation oncedaily for 7 days to patients with CF.

TABLE 17 Loaded Dose: 180 mg Loaded Dose: 240 mg Parameter¹ 50 mg/ml 100mg/ml 100 mg/ml Sputum C_(max) 2,563,119 ± 2,932,121 ± 4,690,808 ±(ng/ml) 1,411,715 (10) 2,559,422 (10) 4,515,727 (10)   Sputum T_(max)(h)    0.27 (10)    0.28 (10)  0.29 (10) Sputum C_(min) 398 ± 482 278 ±192 697 ± 939 (10) (ng/ml) (10) (10) Sputum AUC_((0-t)) 1,889,669 ±1,958,819 ± 4,507,180 ± (h · ng/ml) 1,252,341 (10) 2,109,909 (10)6,593,884 (10)   Sputum AUC_((inf)) 1,890,699 ± 1,960,771 ± 4,517,439 ±(h · ng/ml) 1,252,486 (10) 2,110,392 (10) 6,611,353 (10)   Sputum t½ (h)3.55 ± 2.69 4.34 ± 1.80 4.58 ± 2.54 (10) (10) (10) ¹Arithmetic mean ±standard deviation (N) except for T_(max) for which the median (N) isreported.

There was a 1.6-fold increase in C_(max) between the 180 mg and 240 mgdoses of the 100 mg/ml formulation, of 2,932,121 ng/ml to 4,690,808ng/ml (Table 17). In view of the small number of patients andvariability between subjects, this increase is reasonably consistentwith a predicted increase of about 1.33-fold. In contrast, there was a2.3-fold increase in AUC, from 1,960,771 h·ng/ml [AUC_((inf))] to4,507,180 h·ng/ml [AUC₍₀₋₂₄₎]. The arithmetic mean t½ was comparable forall three treatments, ranging from 3.55 h to 4.58 h (Table 16).

These results show that levofloxacin exposure in sputum was orders ofmagnitude higher than that in serum (Tables 16 and 17). However, theratio of levofloxacin exposure in sputum to that in serum was relativelyindependent of the formulation and the dose, and averaged approximately260,000% for C_(max), and 25,000% for AUC (Table 18).

TABLE 18 Sputum/Serum Ratio Dose: 180 mg Dose: 240 mg Parameter 50 mg/ml100 mg/ml 100 mg/ml C_(max) (ng/ml) 269,336 228,271 274,796 AUC (h ·ng/ml)¹ 23,462 19,911 30,514 ¹AUC_((inf)) for the single 180 mg dosesand AUC_((0-t)) for the multiple 240 mg dose.

Sputum exposure is similar for both formulations. Taking into accountpotential accumulation from the 240 mg QD×7-day regimen, the systemicand sputum exposure after administration of 180 mg and 240 mg as the 100mg/ml formulation appear to be proportional to dose and consistentbetween single and multiple doses.

Table 19 compares levofloxacin AUC and C_(max) results followingnebulization of formulations shown in Examples 4 and 8 as the rawresults or normalized to the RDD or nebulizer loaded dose for eachformulation tested.

TABLE 19 Dose-normalized Sputum Levofloxacin PK Parameters in CFPatients Example Example 4 formulations Example 8 formulationsFormulation/Dose Dose Level C Dose Level Levofloxacin Dose Level C DoseLevel D Dose Level A B (50 mg/ml) with Levofloxacin LevofloxacinLevofloxacin Levofloxacin MgCl₂ and (100 mg/ml) (100 mg/ml) (12 mg/ml)(23.8 mg/ml) Lactose with MgCl₂ with MgCl₂ Loaded Dose 43.3 86.6 180 180240 Estimated RDD 20 40 92 98 131 C_(max) (ng/ml) 86,200 211,5002,563,119 2,932,121 4,690,808 AUC (hr · ng/ml) 67,100 171,400 1,890,6991,960,771 4,517,439 Loaded Dose- 1,991 2,442 14,240 16,290 19,545normalized C_(max) (ng/ml per mg dose) Loaded Dose- 1,550 1,979 10,50410,893 18,823 normalized AUC (hr · ng/ml per mg dose) RDD-normalized4,310 5,288 27,866 29,862 35,830 C_(max) (ng/ml per mg dose)RDD-normalized 3,355 4,285 20,556 19,969 34,505 AUC (hr · ng/ml per mgdose)

The dose-normalized AUC and C_(max) PK parameters show the significantlyincreased exposures of levofloxacin in sputum using the formulations ofExample 8 that include levofloxacin formulated with Mg²⁺ over theformulations of Example 4 that lack Mg²⁺. The differences in sputumconcentrations of levofloxacin between Example 4 and Example 8formulations are further shown in FIG. 14.

Example 9—Mouse Lung Infection Model

A mouse lung infection model was used to compare the efficacy ofintravenous administration with pulmonary administration offluoroquinolones. Eight mice per group were infected with Klebsiellapneumoniae ATCC 43816 by intra-tracheal instillation. Twenty-four hoursafter infection, mice were administered aerosol doses of 10 or 20 mg/kgtwice daily (BID) using a microspray aerosol generation device(PennCentury, Philadelphia, Pa.). Twenty-four hours after beginningtreatment, animals were sacrificed and their lungs were removed,homogenized, and plated to determine colony counts. Table 20 shows theformulations used in this study.

TABLE 20 Levofloxacin in saline Levofloxacin with MgCl₂ Dose (mg/kg) 1020 10 20 Levofloxacin 4 8 4 8 (mg/mL) MgCl₂ (mM) 0 0 8 16 Saline (%) 0.90.9 0 0 Lactose (mM) 0 0 12 24

Levofloxacin formulated with MgCl₂ produced 1 log greater bacterialkilling than levofloxacin formulated in saline at each dose tested (FIG.15). This result is consistent with the increased lung concentrationsdetermined in the rat in Example 2.

Example 10—Efficacy of Aerosol Levofloxacin Formulated with MgCl₂ inMouse Lung Infection Models

This example relates to aerosol administration of levofloxacin withMgCl₂, and intraperiteneal administration of levofloxacin in saline. Thepurpose of the following studies was to determine the efficacy of thesetherapies in acute and chronic lung infection models due to P.aeruginosa.

Antimicrobial agents: Levofloxacin (LKT Laboratories, St. Paul, Minn.),tobramycin (Sicor pharmaceuticals, Irvine, Calif.), and aztreonam (MPBiomedicals, Solon, Ohio) were purchased from independent vendors. Priorto the initiation of each experiment, fresh stock solutions of eachantibiotic were prepared. Levofloxacin formulated with MgCl₂ was dilutedin water; levofloxacin and tobramycin were diluted in 0.9% saline,aztreonam was diluted in 7% sodium bicarbonate in water. Table 21 showsformulations used in this study.

TABLE 21 Levofloxacin in Saline Levofloxacin with MgCl₂ Dose 32 63 12532 63 125 (mg/kg) Levofloxacin 1.5 3 6 6 12 24 (mg/mL) MgCl₂ (mM) 0 0 012 24 48 Saline (%) 0.9 0.9 0.9 0 0 0

Bacterial strains MIC testing: P. aeruginosa ATCC 27853 and NH57388Awere used in these studies. MICs were determined by a brothmicrodilution assay according to CLSI reference methods (Methods fordilution of antimicrobial susceptibility test for bacteria that growaerobically. Seventh Edition: Clinical and Laboratory StandardsInstitute (2006) M&-A7, incorporated by reference in its entirety).Assays were performed in a final volume of 100 μl. The bacterialsuspensions were adjusted to yield a cell density of 5×10⁵ CFU/ml.Antibiotics were prepared at a concentration equivalent to twofold thehighest desired final concentration in culture medium and were thendiluted directly into 96-well microtiter plates. Microtiter plates wereincubated for 24 h at 35° C. and were read by using a microtiter platereader (Molecular Devices) at 600 nm as well as by visual observation byusing a microtiter plate reading mirror. The MIC was defined as thelowest concentration of antibiotic at which the visible growth of theorganism is completely inhibited.

Mice: Female Swiss mice (5-6 wk of age) were obtained from Harlan WestCoast (Germantown, Calif.). All studies were performed under protocolsapproved by an Institutional Animal Care and Use Committee.

Preparation of pseudomonal alginate: P. aeruginosa NH57388A was culturedin 50 ml Mueller-hinton broth (MHB) for 24-28 h at 37° C. with shaking(170 rpm). Bacterial cells were harvested by centrifugation (23,000×g,30 min, at 4° C.) and resuspended in 3-6 ml of MHB. The supernatant wascollected and placed in 80° C. water-bath for 30 min. Alginate wasprecipitated by adding the supernatant to 150 ml of ice-cold 99%ethanol. The precipitated alginate was collected with a sterilebacterial loop and washed several times in sterile saline. The purifiedalginate was then resuspended in 10 ml of sterile saline and stirredvigorously to form a homogeneous suspension. The alginate concentrationwas measured and adjusted to a concentration of 2-3 mg/ml.

Aerosol Administration of antibiotics: Antibiotics were aerosolizedusing a microspray aerosol device (MicroSprayer Model IA-C, PennCentury,Philadelphia, Pa.) attached to a FMJ-250 High-Pressure Syringe(PennCentury, Philadelphia, Pa.). This device produces a 16-22 μM MassMedium Diameter spray. For administration, each mouse was anesthetized(5% isoflurane in oxygen running at 4 L/min) and positioned securely ata 45-50° angle by the upper teeth, the microspray aerosol tip wasinserted to the bifurcation and a 50 μl volume was administered.

Pharmacokinetics: Mice (n=3/timepoint) were administered single 60 mg/kgaerosol dose of levofloxacin formulated with MgCl₂ or a 20 mg/kg IP doseof levofloxacin. Mice were sacrificed at 0.08, 0.16, 0.25, 0.5, 0.75,1.0, 2.0, 3.0, and 4.0 h after dosing and their lungs collected.Levofloxacin lung homogenate concentrations administered as levofloxacinor levofloxacin formulated with MgCl₂ were measured using an HPLCmethod. Analytical standards (0.05 to 100 mg/L) were prepared in freshmouse lung homogenate collected from untreated animals. Lung homogenateor standards for both compounds were mixed with double the volume of 4%trichloroacetic acid, vortexed and then centrifuged at 12,000 rpm for 10min using a refrigerated Eppendorf 5415c centrifuge set at 4-10° C.Aliquots of the supernatant (25 μl) were injected directly onto the HPLCusing a temperature-controlled autoinjector set at 10° C. Standardcurves were constructed of the peak area versus standard concentration,and the data were fit using weighted linear regression (Microsoft Excel,Seattle, Wash.). The concentrations of levofloxacin in the lunghomogenate were calculated from these standard curves. The lungpharmacokinetic parameters were determined using WinNonlin (Pharsight,Mountain View, Calif.).

Acute Mouse Lung Infection Model: P. aeruginosa ATCC 27853 was grownovernight in MHB at 35° C. The bacterial suspensions were adjusted toapproximately 1-6×10⁵ CFU/ml by correlation of the absorbance at 600 nmwith predetermined plate counts. Female Swiss mice were made neutropenicby the intraperitoneal (IP) injection of 150 mg/kg cyclophosphamide(Baxter, Deerfield) on days 1 and 3. On day 4, mice were infected byintratracheal (IT) instillation of 0.05 ml of inoculum using a curvedoral gavage tip attached to a 1 ml syringe. Antibiotic treatmentsstarted 24 h post-infection and were administered once or twice dailyfor 24 or 48 h. Antibiotics were aerosolized using a microspray aerosoldevice. All infections and aerosol treatments were performed underisoflurane anesthesia (5% isoflurane in oxygen running at 4 L/min). Anuntreated group of mice (n=8) was sacrificed prior to the initiation oftreatment to determine baseline bacterial counts. The treated animals(n=8) were sacrificed 12-16 h following the last antibiotic dose bycarbon dioxide asphyxiation. The lungs were removed aseptically andhomogenized (Pro200 homogenizer, Pro Scientific, Monroe, Conn.) in 1 mlof sterile saline. Serial 10-fold dilutions of the homogenized lung wereplated on Mueller-hinton agar (MHA), and colonies counted. For survivalstudies, mice (n=10) were observed for 7 days after the end of treatmentor a total of 9 days post-infection.

Chronic Mouse Lung Infection Model: P. aeruginosa NH57388A was culturedin 50 ml MHB for 24-28 h at 37° C. with shaking (170 rpm). Bacterialcells were harvested by centrifugation (23,000×g, 30 min, at 4° C.) andresuspended in 3-6 ml of MHB (Hoffmann, N. T. B. et al. 2005. Novelmouse model of chronic Pseudomonas aeruginosa lung infection mimickingcystic fibrosis. Infect Immun 73:2504-14, incorporated herein byreference in its entirety). The bacterial suspension was diluted (1:10)in the alginate suspension to yield about 10⁸ CFU/ml. Initialestablishment of infection was achieved by a transient neutropenia usinga single 150 mg/kg IP dose of cyclophosphamide 4 days prior toinfection. On day 4, the mice were infected using a curved bead-tippedoral gavage attached to a 1 ml syringe while under isofluraneanesthesia. Antibiotic treatments started 24 h post-infection and wereadministered twice daily for three consecutive days with variousconcentrations of antibiotics either by the IP route or by aerosol usinga microspray device. 12-16 h following the last treatment, mice weresacrificed and colony counts in the lung determined as described herein.

Statistical Analysis: Survival and lung bacterial counts were analyzedby log-rank and the Mann-Whitney U test (GraphPad Prism version of4.03), respectively. A P value of <0.05 was considered statisticallysignificant.

Minimal Inhibitory Concentration of Antibiotics

The minimal inhibitory concentration (MIC) of the P. aeruginosa strainsused in animal studies are shown in Table 22. Tobramycin was the mostpotent antibiotic in vitro, with MICs of <1 μg/ml, levofloxacinformulated with MgCl₂ and levofloxacin had MICs of 1 and 2 μg/ml, andaztreonam had MICs of 4 μg/ml against both strains

TABLE 22 MIC (μg/ml) Levofloxacin P. aeruginosa formulated with strainMgCl₂ Levofloxacin Tobramycin Aztreonam ATCC27853 1 1 0.25 4 NIH57388ACF 2 2 0.5 4

Mouse Pharmacokinetics

Normalized lung pharmacokinetic parameters for levofloxacin formulatedwith MgCl₂ and levofloxacin are shown in Table 23. Aerosoladministration of 60 mg/kg levofloxacin formulated with MgCl₂ producedvalues for levofloxacin AUC and C_(max) that were 9 and 30-fold higherthan those achieved with dose normalized intraperitoneal administrationof levofloxacin.

TABLE 23 Levofloxacin formulated Levofloxacin Parameter with MgCl₂formulated in Saline Route of Administration Aerosol IP Dose (mg/kg) 6020 C_(max) (mg/kg) 550 6.2 (18.6) (Normalized to 60 mg/kg) AUC (hr ·mg/kg) 106 4.1 (12.3) (Normalized to 60 mg/kg)Aerosol Levofloxacin Formulated with MgCl₂ vs. Systemic Levofloxacin inAcute and Chronic Lung Infection Models

In the acute lung infection model, aerosol treatment with 125, 62.5, and32 mg/kg of levofloxacin formulated with MgCl₂ produced 5.9, 4.3, and2.3 log CFU reductions in lung bacterial counts, respectively (FIG. 16).Systemic treatment with 125, 62.5, and 32 mg/kg of levofloxacin produced3.5, 2.7, and 0.65 log CFU reductions, respectively. The reduction inbacterial counts with aerosol levofloxacin formulated with MgCl₂ wasgreater than that observed with IP levofloxacin on a per dose basis(p<0.05).

In the chronic lung infection model, intraperitoneal treatment with 60,30, and 15 mg/kg of levofloxacin in saline produced a 0.15, 0.32, and0.83 log increase in bacterial counts, respectively (FIG. 17). Incontrast, aerosol dosing with 60, 30, and 15 mg/kg of levofloxacinformulated with MgCl₂ produced 1.26, 0.62, and 0.07 log decreases inbacterial counts, respectively. Overall, bacterial load in the lung wassignificantly lower in mice treated with aerosolized levofloxacinformulated with MgCl₂ compared to systemic levofloxacin on a dose perdose basis in both infection models (p<0.05 for levofloxacin formulatedwith MgCl₂ vs. systemic levofloxacin).

Aerosol Levofloxacin, Tobramycin, and Aztreonam in an Acute Lethal LungInfection Model

To compare the effects of levofloxacin formulated with MgCl₂,tobramycin, and aztreonam in the acute lung infection model, mice wereinfected with P. aeruginosa ATCC 27853 and treated by the aerosol routetwice a day for 2 consecutive days. Due to toxicity, tobramycin waslimited to a 60 mg/kg maximum dose and aztreonam was limited to 400mg/kg maximum dose. In addition, due to the need for anesthesia fortreatment, the maximum number of daily doses was limited to two.

As shown in FIG. 18, aerosol dosing with levofloxacin formulated withMgCl₂, tobramycin, and aztreonam produced mean reductions of 4.10, 2.70,and 0.24 log CFU per lung, respectively (p<0.05 for comparisons oflevofloxacin formulated with MgCl₂ with aztreonam). Notably,administration of the same total daily dose of levofloxacin formulatedwith MgCl₂ as single or twice daily doses resulted in similar reductionsin P. aeruginosa counts in the lung.

Survival was monitored over 9 days. As shown in FIG. 19, all untreatedmice succumbed to the infection after 3 days. Treatment with 800mg/kg/day (400 mg/kg BID) aerosolized aztreonam had the lowest survivalrate among the antibiotics used in this study (20%) and was notsignificantly different from untreated mice (p>0.05). Treatment with 120mg/kg/day (60 mg/kg BID) tobramycin produced a 60% survival rate whichwas statistically different than controls (p<0.05). Treatment with 120mg/kg/day levofloxacin formulated with MgCl₂ as either 120 mg/kg QD or60 mg/kg BID produced 100% survival which was significantly differentfrom untreated controls or aztreonam (p<0.05), but not significantlydifferent from tobramycin (p=0.056).

Aerosol Levofloxacin, Tobramycin, and Aztreonam in a Chronic LungInfection Model

Aerosolized levofloxacin formulated with MgCl₂, tobramycin and aztreonamproduced mean log CFU reductions of 3.3, 2.9, and 1.25, respectively(FIG. 20). Aerosolized doses of either tobramycin or levofloxacinformulated with MgCl₂ produced significantly lower bacterial countscompared to aztreonam, or untreated control groups (p<0.05).

These in vivo studies show that aerosol dosing of levofloxacinformulated with MgCl₂ produces greater antibacterial killing thansystemic dosing in both acute and chronic P. aeruginosa lung infectionmodels. Notably, twice daily dosing with levofloxacin formulated withMgCl₂ reduced the lung bacterial load by an extent similar to or greaterthan that observed with aerosolized tobramycin and aztreonam (FIG. 18).This reduction in bacterial load in the lungs translated to improvedsurvival (FIG. 19).

In addition, comparisons of single-versus twice-daily dosing oflevofloxacin formulated with MgCl₂ showed comparable bacterial killingand survival, suggesting that once-daily treatment with levofloxacinformulated with MgCl₂ may be possible in patients. Once dailyadministration of a medicament is particularly advantageous overmultiple administrations, where multiple administrations areinconvenient to patients and can result in poor adherence to treatment.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will occur to those skilled inthe art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

1.-10. (canceled)
 11. A method of treating a chronic pulmonary infectiondue to Pseudomonas aeruginosa in a subject with cystic fibrosis in needthereof, the method comprising administering to the lungs of the subjectwith cystic fibrosis about 240 mg of an aerosol of a solution comprisingabout 100 mg/ml of levofloxacin and about 200 mM of magnesium chloride;wherein the solution has a pH from about 5 to about 7 and an osmolalityfrom about 350 mOsmol/kg to about 400 mOsmol/kg, to treat the chronicpulmonary infection due to Pseudomonas aeruginosa.
 12. A method oftreating a Pseudomonas aeruginosa pulmonary infection in a subject inneed thereof, the method comprising administering to the lungs of thesubject an aerosol of a solution comprising from about 90 mg/ml to about110 mg/ml of levofloxacin and from about 190 mM to about 210 mM of amagnesium cation to treat the Pseudomonas aeruginosa pulmonaryinfection.
 13. The method of claim 12, wherein the subject has cysticfibrosis.
 14. The method of claim 12, wherein the solution comprisesabout 100 mg/ml of levofloxacin and about 200 mM of the magnesiumcation; wherein the solution has a pH from about 5 to about 7 and anosmolality from about 300 mOsmol/kg to about 500 mOsmol/kg; and whereinfrom about 200 mg to about 280 mg of levofloxacin is administered to thesubject.
 15. The method of claim 14 wherein from about 230 mg to about250 mg of levofloxacin is administered to the subject.
 16. The method ofclaim 14 wherein the solution has a pH from about 6.0 to about 6.5 andan osmolality from about 350 mOsmol/kg to about 400 mOsmol/kg.
 17. Themethod of claim 14, wherein the magnesium cation is in the form ofmagnesium chloride.